Recombinant aavs having useful transcytosis properties

ABSTRACT

The disclosure in some aspects relates to recombinant adeno-associated viruses having distinct tissue targeting capabilities. In some aspects, the disclosure relates to gene transfer methods using the recombinant adeno-associate viruses. In some aspects, the disclosure relates to isolated AAV capsid proteins and isolated nucleic acids encoding the same.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/058,938, filed Aug. 8, 2018, entitled “RECOMBINANT AAVS HAVING USEFULTRANSCYTOSIS PROPERTIES”, which is a continuation of U.S. applicationSer. No. 15/120,294, filed Aug. 19, 2016, entitled “RECOMBINANT AAVSHAVING USEFUL TRANSCYTOSIS PROPERTIES”, which is a national stage filingunder 35 U.S.C. § 371 of International Patent Application Serial No.PCT/US2015/016691, filed Feb. 19, 2015, and entitled “RECOMBINANT AAVSHAVING USEFUL TRANSCYTOSIS PROPERTIES”, which claims the benefit under35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No. 61/942,002,entitled “RECOMBINANT AAVS HAVING USEFUL TRANSCYTOSIS PROPERTIES”, filedon Feb. 19, 2014, the entire contents of each of which are incorporatedby reference herein.

FIELD

The disclosure in some aspects relates to isolated nucleic acids,compositions, and kits is useful for identifying adeno-associatedviruses in cells. In some aspects, the disclosure provides novel AAVsand methods of use thereof as well as related kits.

BACKGROUND

Adeno-associated virus (AAV) is a small (·26 nm) replication-defective,nonenveloped virus, that depends on the presence of a second virus, suchas adenovirus or herpes virus, for its growth in cells. AAV wasdiscovered in 1960s as a contaminant in adenovirus (a cold causingvirus) preparations. Its growth in cells is dependent on the presence ofadenovirus and, therefore, it was named as adeno-associated virus. AAVcan infect both dividing and non-dividing cells and may incorporate itsgenome into that of the host cell. These features make AAV a veryattractive candidate for creating viral vectors for gene therapy.

SUMMARY

The disclosure in some aspects relates to novel AAVs for gene therapyapplications. Aspects of the disclosure relate to variants of AAV9 thathave unique transcytosis properties. In some embodiments, recombinantAAVs (rAAVs) disclosed herein have a transduction efficiency similar torAAV9 but improved safety profiles, particularly for muscle and lunggene delivery. Accordingly, in some embodiments, rAAV vectors areparticularly useful for gene therapy of muscular, lung or otherdisorders. In some embodiments, a structural analysis of the ClvD8capsid protein (a variant of the AAV9 capsid) was performed using therecently established AAV9 capsid structure as a template.

In some embodiments, this analysis revealed differences in amino acidsat position 647 and in other residues located at a positioncorresponding to AAV9 capsid protrusions that surrounds the icosahedral3-fold axis of AAV9 VP3 protein. In some embodiments, amino acids inthese protrusions play a role in receptor binding and cellulartransduction. In some embodiments, amino acids involved in peripheral(primarily liver) tissue transduction by IV delivery of rAAV9 wereidentified. In some embodiments, single and combinatory mutations foreach of the amino acids on the AAV9 capsid that are mutated in AAV-ClvD8were produced and evaluated for biodistribution profiles followingdelivery by intranasal or intramuscular injection. In some embodiments,a novel capsid mutant of AAV9, referred to as AAV9HR, produced localtissue-restricted expression and genome persistence by intramuscular andintranasal delivery, and produced low levels of expression in liver whendelivered by intravascular (IV), intramuscular (IM) and intranasal (IN)delivery routes. In some embodiments, AAV9.HR displayed a bloodclearance pattern similar to that of rAAV9wt after IV, IM, and INadministration indicating that its ability to cross vascular barrierfrom tissue to vessel was not impaired.

Accordingly, aspects of the disclosure relate to recombinant AAVs(rAAVs) comprising an AAV capsid protein having an amino acid sequenceselected from the group consisting of: SEQ ID NOs: 2-7, and compositionscomprising the same. In some embodiments, compositions provided hereinthat comprise an rAAV further comprise a pharmaceutically acceptablecarrier. In some embodiments, host cells are provided that contain anucleic acid that comprises a coding sequence selected from the groupconsisting of: SEQ ID NO: 9-14 that is operably linked to a promoter,such that the cells are engineered to express rAAV capsids. In someembodiments, compositions are provided that comprise a host cell and asterile cell culture medium. In some embodiments, the compositionsfurther comprise a cryopreservative.

Aspects of the disclosure relate to methods for delivering a transgeneto a subject. In some embodiments, the methods involve administering arAAV to a subject, in which the rAAV comprises: (i) a capsid proteinhaving a sequence selected from SEQ ID NOs: 9 to 14, and (ii) at leastone transgene, and in which the rAAV infects cells of a target tissue ofthe subject. In some embodiments, the target tissue is skeletal muscle,heart, liver, pancreas, brain or lung. In some embodiments, the rAAV isadministered intravenously, transdermally, intraocularly, intrathecally,orally, intramuscularly, subcutaneously, intranasally, or by inhalationto a subject. In some embodiments, the subject is selected from a mouse,a rat, a rabbit, a dog, a cat, a sheep, a pig, and a non-human primate.In some embodiments, the subject is a human.

In some embodiments, at least one transgene of an rAAV encodes aprotein. In some embodiments, the protein is an an antigen-bindingprotein, such as an immunoglobulin heavy chain or light chain, orfragment of either one. In some embodiments, the protein is a singlechain Fv fragment or Fv-Fc fragment. Accordingly, in some embodiments,the rAAV can be used to infect cells of target tissue (e.g., muscletissue) so as to engineer cells of the tissue to express anantigen-binding protein, such as an antibody or fragment thereof. Insome embodiments, the protein is a central nervous system (CNS) protein.In some embodiments, the CNS protein is aspartoacylase (ASPA). In someembodiments, the ASPA is human ASPA. Accordingly, in some embodiments,the rAAV can be used to infect cells of a target tissue (e.g., CNStissue) so as to engineer cells of the tissue to express ASPA.

In some embodiments, the protein is a secreted tumor suppressor protein.In some embodiments, the secreted tumor suppressor protein is selectedfrom the group consisting of IGFBP7 and SRPX.

In other embodiments, the at least one transgene of an rAAV encodes asmall interfering nucleic acid, such as a miRNA.

In some embodiments, the transgene comprises one or more elements thatmediate tissue-specific expression. In some embodiments, the transgeneof an rAAV expresses a transcript that comprises at least one bindingsite for a miRNA, wherein the miRNA inhibits activity of the transgene,in a tissue other than the target tissue, by hybridizing to the bindingsite. In some embodiments, the transgene of an rAAV comprises a tissuespecific promoter or inducible promoter. In some embodiments, the tissuespecific promoter is a liver-specific thyroxin binding globulin (TBG)promoter, a insulin promoter, a glucagon promoter, a somatostatinpromoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn)promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES)promoter, a α-myosin heavy chain (a-MHC) promoter, or a cardiac TroponinT (cTnT) promoter.

Aspects of the disclosure relate to isolated nucleic acids encoding anAAV caspid protein having an amino acid sequence selected from the groupconsisting of: SEQ NOs: 2-7. In some embodiments, isolated nucleic acidsare provided that comprise a sequence selected from the group consistingof: SEQ ID NO: 9-14. In some embodiments,

Aspects of the disclosure relate to isolated AAV capsid proteincomprising an amino acid sequence selected from the group consisting of:SEQ ID NOs: 2-7 and compositions comprising the same.

Aspects of the disclosure relate to kits for producing a rAAV. In someembodiments, the kits comprise a container housing an isolated nucleicacid having a sequence of any one of SEQ ID NO: 9-14. In someembodiments, the kits further comprise at least one container housing arecombinant AAV vector, wherein the recombinant AAV vector comprises atransgene. In some embodiments, the kits comprise a container housing arecombinant AAV having an isolated AAV capsid protein having an aminoacid sequence as set forth in any of SEQ ID NOS: 2-7. In someembodiments, the kits further comprise instructions for producing therAAV.

Aspects of the disclosure relate to methods of delivering rAAVs for thetreatment of disease. In some embodiments, the disease is a centralnervous system ((CNS) disease. In some embodiments, the CNS disease isCanavan disease. Accordingly, in some embodiments the disclosureprovides a method for treating Canavan disease, the method comprisingadministering to a subject a therapeutically effective amount of a rAAVto a subject, wherein the rAAV comprises (i) a capsid protein having asequence selected from SEQ ID NOs: 10 to 14, and (ii) at least onetransgene, wherein the at least one transgene encodes ASPA; and whereinthe rAAV infects cells of a target tissue of the subject.

In some embodiments of the method, the target tissue is CNS tissue. Insome embodiments, the ASPA is human ASPA.

In some embodiments, the disease is cancer. Accordingly, in someembodiments, the disclosure provides a method for treating cancer, themethod comprising administering to a subject a therapeutically effectiveamount of a rAAV to a subject, wherein the rAAV comprises: (i) a capsidprotein having a sequence, selected from SEQ NOs: 10 to 14, and (ii) atleast one transgene, wherein the at least one transgene encodes a tumorsuppressor protein; and wherein the rAAV infects cells of a targettissue of the subject.

In some embodiments, the tumor suppressor protein is selected fromthe)up consisting of IGFBP7 and SRPX.

Each of the limitations of the disclosure can encompass variousembodiments of the disclosure. It is, therefore, anticipated that eachof the limitations of the disclosure involving any one element orcombinations of elements can be included in each aspect of thedisclosure. This disclosure is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The disclosureis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled to in everydrawing. In the drawings:

FIGS. 1A-1C depict an alignment of AAV9 variants; sequences in thealignment are as follows: AAV9: SEQ ID NO:1, CLvD8: SEQ ID NO: 2, AAV9Y:SEQ ID NO: 6, AAV9H: SEQ ID NO: 3, AAV9R: SEQ ID NO: 5, AAV9i: SEQ IDNO: 4, AAV.HR: SEQ ID NO: 7, Consensus: SEQ ID NO: 15;

FIGS. 2A-2D depict results showing that Clv-D8 has reduced transcytosiscapability compared with AAV9;

FIG. 3 depicts that AAV9.HR (derived from CLv-D8, Δ2 a.a.) shows similarblood clearance pattern compared to wt AAV9 after IV, IN and IMinjection;

FIGS. 4A-4C depict that AAV9.HR (derived from CLv-D8, Δ2 a.a .)-mediatedtransgene expression is limited to injected muscle after IM injection;

FIG. 5 depicts that AAV9.HR (derived from CLv-D8, Δ2 a.a.)-mediatedliver transgene expression is significantly lower than wtAAV9 after IVinjection;

FIG. 6 depicts that AAV9.HR (derived from CLv-D8, Δ2 a.a.)-mediatedtransgene expression is limited to lung after IN delivery;

FIG. 7 depicts results of assays in which six-week old male C57BL/6 micewere injected via tail vein (1.0 e11 GC/mouse) with AAV9wt and AAV9.HRencoding luciferase gene; four weeks later, specific organs wereharvested for qPCR analysis;

FIG. 8 depicts results of assays in which six-week old male C57BL/6 micewere injected via tail vein (1.0 e11 GC/mouse) with AAV9wt and AAV9.HRencoding luciferase gene; four weeks later, specific organs wereprocessed for luciferase activity analysis;

FIG. 9 depicts results of assays in which four-week-old C57BL/6 micewere injected (IV, Retro-orbital injection, 1e12 GC/mouse,) withscAAV9wt.CB6.PI.EGFP (n=3) or scAAV9.HR.CB6.PI.EGFP (n=3); three weeksafter administration, organs were collected and gDNA were extracted forqPCR analysis (Student's t-test (unpaired, two-tailed));

FIG. 10 depicts a comparison of AAV9 and AAV9.HR-mediated neurontransduction after IV (RO) delivery into D1 mice at 4×10¹¹ GC/mouse at 3weeks post-injection;

FIG. 11 depicts AAV9.HR mediated EGFP expression in brain after IV (RO)delivery into D1 mice at 4×10¹¹ GC/mouse at 3 weeks post-injection;

FIG. 12 depicts bio-distribution of AAV9 and AAV9.HR in adult mice (HighDose, eGFP);

FIGS. 13A-13B depict hAspA enzyme expression (western blot) and activityafter intravenous injection;

FIGS. 14A-14B depict MRI and MRS of mice at P42 after intravenousinjection of AAV9 and AAV9.HR constructs. WT: wild-type; CD: Canavandisease; FKzhAspA-Opt: full kozak human AspA/AAV9 construct;HR.FKzhAspA-Opt: full kozak human AspA/AAV9.HR construct;

FIGS. 15A-15B depict weight and age of mice treated with vehicle (NaCl),FKzhAspA-Opt or HR.FKzhAspA-Opt by intravenous injection. Untreated CDmice died at 4 weeks of age;

FIG. 16 depicts rotarod assessment of mice treated with vehicle (:NaCl),FKzhAspA-Opt or HR.FKzhAspA-Opt by intravenous injection;

FIG. 17 depicts inverted screen assessment of mice treated with vehicle(NaCl), FKzhAspA-Opt or HR.FKzhAspA-Opt by intravenous injection;

FIG. 18 depicts balance beam assessment of mice treated with vehicle(NaCl), FKzhAspA-Opt or HR.FKzhAspA-Opt by intravenous injection;

FIGS. 19A-19B depict AAV.HR-IGEBP7 delivery by intramuscular injectionleads to tumor suppression in a dose dependent manner;

FIGS. 20A-20B depict a dose curve for AAV.HR-SRPX induced tumorsuppression in Balb/c Nu/Nu mice;

FIG. 21 depicts reversibility of AAV-mediated delivery of transgene;

FIG. 22 depicts AAV genomes are restricted in injection site afterdelivery of AAV.HR vectors;

FIG. 23 depicts a toxicity assay for AAVHR.SRPXwt; ALT: Alaninetransaminase; AST: aspartate aminotransferase;

FIG. 24 depicts AST level at each time point (2-weeks to 12-weekspost-injection) of the toxicity assay;

FIG. 25 depicts ALT level at each time point (2-weeks to 12-weekspost-injection) of the toxicity assay.

DETAILED DESCRIPTION

In some embodiments, recombinant AAVs are provided herein that overcomeshortcomings of existing rAAV technologies in that they have distincttranscytosis properties that make them useful for certain gene therapyand research applications.

In some aspects of the disclosure new AAV capsid proteins are providedthat have distinct tissue targeting capabilities. In some embodiments,an AAV capsid protein is isolated from the tissue to which an AAVcomprising the capsid protein targets. In some aspects, methods fordelivering a transgene to a target tissue in a subject are provided. Thetransgene delivery methods may be used for gene therapy (e.g., to treatdisease) or research (e.g., to create a somatic transgenic animal model)applications.

Isolated AAV Capsid Proteins and Nucleic Acids Encoding the Same

AAVs are natural inhabitants in mammals. AAVs isolated from mammals,particularly non-human primates, are useful for creating gene transfervectors for clinical development and human gene therapy applications.The disclosure provides in some aspects novel AAV capsid proteins thathave been development through functional mutagenesis. Protein and aminoacid sequences as well as other information regarding the AAVs capsidare set forth in the sequence listing.

In some embodiments, an AAV capsid is provided that has an amino acidsequence of SEQ ID NO: 1 with one or more of the following amino acidalterations: Y445H, H527Y, I647T, and R533S.

In some embodiments, an AAV capsid is provided that has an amino acidsequence of SEQ ID NO: 1 with one or more of the following amino acidalterations: Y445H, H527Y, I647T, and R533S and up to 5, up to 10, up to20, up to 30, up to 40, up to 50, up to 100 other amino acidalternations (e.g., conservative amino acid substitutions).

An example of an isolated nucleic acid that encodes an AAV capsidprotein is a nucleic acid having a sequence, selected from the, groupconsisting of: SEQ ID NO: 9-14 as well as nucleic acids havingsubstantial homology thereto. In some embodiments, isolated nucleicacids that encode AAV capsids are provided that have sequences selectedfrom: SEQ ID NO: 9, 10, 11, 12, 13, and 14.

In some embodiments, nucleic acids are provided that encode an AAVcapsid having one or more of the following amino acid alterations:Y445H, H527Y, I647T, and R533S based on the amino acid sequence of SEQID NO: 1.

In some embodiments, nucleic acids are provided that encode an AAVcapsid having one or more of the following amino acid alterations:Y445H, H527Y, I647T, and R533S based on the amino acid sequence of SEQID NO: 1 and up to 5, up to 10, up to 20, up to 30, up to 40, up to 50,up to 100 other amino acid alternations.

In some embodiments, a fragment (portion) of an isolated nucleic acidencoding a AAV capsid sequence may be useful for constructing a nucleicacid encoding a desired capsid sequence. Fragments may be of anyappropriate length (e.g., at least 9, at least 18, at least 36, at least72, at least 144, at least 288, at least 576, at least 1152 or morenucleotides in length). The fragment may be a fragment that does notencode a peptide that is identical to a sequence of SEQ ID NO: 1. Forexample, a fragment of nucleic acid sequence encoding a variant aminoacid (compared with a known AAV serotype) may be used to construct, ormay be incorporated within, a nucleic acid sequence encoding an AAVcapsid sequence to alter the properties of the AAV capsid. For example,a nucleic sequence encoding an AAV variant may comprise n amino acidvariants (e.g., in which n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)compared with a known AAV serotype (e.g., AAV9). A recombinant capsequence may be constructed having one or more of the n amino acidvariants by incorporating fragments of a nucleic acid sequencecomprising a region encoding a variant amino acid into the sequence of anucleic acid encoding the known AAV serotype. The fragments may beincorporated by any appropriate method, including using site directedmutagenesis. Thus, new AAV variants may be created having newproperties.

In some cases, fragments of capsid proteins disclosed herein areprovided. Such fragments may at least 10, at least 20, at least 50, atleast 100, at least 200, at least 300, at least 400, at least 500 ormore amino acids in length. In some embodiments, chimeric capsidproteins are provided that comprise one or more fragments of one or morecapsid proteins disclosed herein.

“Homology” refers to the percent identity between two polynucleotides ortwo polypeptide moieties. The term “substantial homology”, whenreferring to a nucleic acid, or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in about 90 to 100% of the alignedsequences. When referring to a polypeptide, or fragment thereof, theterm “substantial homology” indicates that, when optimally aligned withappropriate gaps, insertions or deletions with another polypeptide,there is nucleotide sequence identity in about 90 to 100% of the alignedsequences. The term “highly conserved” means at least 80% identity,preferably at least 90% identity, and more preferably, over 97%identity. In some cases, highly conserved may refer to 100% identity.Identity is readily determined by one of skill in the art by, forexample, the use of algorithms and computer programs known by those ofskill in the art.

As described herein, alignments between sequences of nucleic acids orpolypeptides are performed using any of a variety of publicly orcommercially available Multiple Sequence Alignment Programs, such as“Clustal W”, accessible through Web Servers on the Internet.Alternatively, Vector NTI utilities may also be used. There are also anumber of algorithms known in the art which can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using BLASTN, which provides alignments and percent sequenceidentity of the regions of the best overlap between the query and searchsequences. Similar programs are available for the comparison of aminoacid sequences, e.g., the “Clustal X” program, BLASTP. Typically, any ofthese programs are used at default settings, although one of skill inthe art can alter these settings as needed. Alternatively, one of skillin the art can utilize another algorithm or computer program whichprovides at least the level of identity or alignment as that provided bythe referenced algorithms and programs. Alignments may be used toidentify corresponding amino acids between two proteins or peptides. A“corresponding amino acid” is an amino acid of a protein or peptidesequence that has been aligned with an amino acid of another protein orpeptide sequence. Corresponding amino acids may be identical ornon-identical. A corresponding amino acid that is a non-identical aminoacid may be referred to as a variant amino acid.

Alternatively for nucleic acids, homology can be determined byhybridization of polynucleotides under conditions which form stableduplexes between homologous regions, followed by digestion withsingle-stranded-specific nuclease(s), and size determination of thedigested fragments. DNA sequences that are substantially homologous canbe identified in a Southern hybridization experiment under, for example,stringent conditions, as defined for that particular system. Definingappropriate hybridization conditions is within the skill of the art.

A “nucleic acid” sequence refers to a DNA or RNA sequence. In someembodiments, proteins and nucleic acids of the disclosure are isolated.As used herein, the term “isolated” means artificially produced. As usedherein with respect to nucleic acids, the term “isolated” means: (i)amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulableby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulable by standard techniques known to those of ordinary skill inthe art. As used herein with respect to proteins or peptides, the term“isolated” refers to a protein or peptide that has been isolated fromits natural environment or artificially produced (e.g., by chemicalsynthesis, by recombinant DNA technology, etc.).

The skilled artisan will also realize that conservative amino acidsubstitutions may be made to provide functionally equivalent variants,or homologs of the capsid proteins. In some aspects the disclosureembraces sequence alterations that result in conservative amino acidsubstitutions. As used herein, a conservative amino acid substitutionrefers to an amino acid substitution that does not alter the relativecharge or size characteristics of the protein in which the amino acidsubstitution is made. Variants can be prepared according to methods foraltering polypeptide sequence known to one of ordinary skill in the artsuch as are found in references that compile such methods, e.g.Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,New York, 1989, or Current. Protocols in Molecular Biology, F. M.Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservativesubstitutions of amino acids include substitutions made among aminoacids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K,R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Therefore, one canmake conservative amino acid substitutions to the amino acid sequence ofthe proteins and polypeptides disclosed herein.

Recombinant AAVs

In some aspects, the disclosure provides isolated AAVs. As used hereinwith respect to AAVs, the term “isolated” refers to an AAV that has beenisolated from its natural environment (e.g., from a host cell, tissue,or subject) or artificially produced. Isolated AAV's may be producedusing recombinant methods. Such AAVs are referred to herein as“recombinant AAVs”. Recombinant AAVs (rAAVs) preferably havetissue-specific targeting capabilities, such that a transgene of therAAV will be delivered specifically to one or more predeterminedtissue(s). The AAV capsid is an important element in determining thesetissue-specific targeting capabilities. Thus, an rAAV having a capsidappropriate for the tissue being targeted can be selected. In someembodiments, the rAAV comprises a capsid protein having an amino acidsequence as set forth in any one of SEQ NOs 2-7, or a protein havingsubstantial homology thereto.

Methods for obtaining recombinant AAVs having a desired capsid proteinare well known in the art. (See, for example, US 2003/0138772), thecontents of which are incorporated herein by reference in theirentirety). Typically the methods involve culturing a host cell whichcontains a nucleic acid sequence encoding an AAV capsid protein (e.g., anucleic acid having a sequence as set forth in any one of SEQ ID NOs9-14) or fragment thereof; a functional rep gene; a recombinant AAVvector composed of, AAV inverted terminal repeats (ITRs) and atransgene; and sufficient helper functions to permit packaging of therecombinant AAV vector into the AAV capsid proteins.

The components to be cultured in the host cell to package a rAAV vectorin an AAV capsid may be provided to the host cell in trans.Alternatively, any one or more of the required components (e.g.,recombinant AAV vector, rep sequences, cap sequences, and/or helperfunctions) may be provided by a stable host cell which has beenengineered to contain one or more of the required components usingmethods known to those of skill in the art. Most suitably, such a stablehost cell will contain the required component(s) under the control of aninducible promoter. However, the required component(s) may be under thecontrol of a constitutive promoter. Examples of suitable inducible andconstitutive promoters are provided herein, in the discussion ofregulatory elements suitable for use with the transgene. In stillanother alternative, a selected stable host cell may contain selectedcomponent(s) under the control of a constitutive promoter and otherselected component(s) under the control of one or more induciblepromoters. For example, a stable host cell may be generated which isderived from 293 cells (which contain E1 helper functions under thecontrol of a constitutive promoter), but which contain the rep and/orcap proteins under the control of inducible promoters. Still otherstable host cells may be generated by one of skill in the art.

The recombinant AAV vector, rep sequences, cap sequences, and helperfunctions required for producing the rAAV of the disclosure may bedelivered to the packaging host cell using any appropriate geneticelement (vector). The selected genetic element may be delivered by anysuitable method, including those described herein. The methods used toconstruct any embodiment of this disclosure are known to those withskill in nucleic acid manipulation and include genetic engineering,recombinant engineering, and synthetic techniques. See, e.g., Sambrooket al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virionsare well known and the selection of a suitable method is not alimitation on the present disclosure. See, e.g., K. Fisher et al, J.Virol, 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

In some embodiments, recombinant AAVs may be produced using the tripletransfection method (described in detail in U.S. Pat. No. 6,001,650).Typically, the recombinant AAVs are produced by transfecting a host cellwith an recombinant AAV vector (comprising a transgene) to be packagedinto AAV particles, an AAV helper function vector, and an accessoryfunction vector. An AAV helper function vector encodes the “AAV helperfunction” sequences (i.e., rep and cap), which function in trans forproductive AAV replication and encapsidation. Preferably, the AAV helperfunction vector supports efficient AAV vector production withoutgenerating any detectable wild-type AAV virions (i.e., AAV virionscontaining functional rep and cap genes). Non-limiting examples ofvectors suitable for use with the present disclosure include pHLP19,described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described inU.S. Pat. No. 6,156,303, the entirety of both incorporated by referenceherein. The accessory function vector encodes nucleotide sequences fornon-AAV derived viral and/or cellular functions upon which AAV isdependent for replication (i.e., “accessory functions”). The accessoryfunctions include those functions required for AAV replication,including, without limitation, those moieties involved in activation ofAAV gene transcription, stage specific AAV mRNA splicing, AAV DNAreplication, synthesis of cap expression products, and AAV capsidassembly. Viral-based accessory functions can be derived from any of theknown helper viruses such as adenovirus, herpesvirus (other than herpessimplex virus type-1), and vaccinia virus.

In some aspects, the disclosure provides transfected host cells. Theterm “transfection” is used to refer to the uptake of foreign DNA by acell, and a cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al, (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousnucleic acids, such as a nucleotide integration vector and other nucleicacid molecules, into suitable host cells.

A “host cell” refers to any cell that harbors, or is capable ofharboring, a substance of interest. Often a host cell is a mammaliancell. A host cell may be used as a recipient of an AAV helper construct,an AAV minigene plasmid, an accessory function vector, or other transferDNA associated with the production of recombinant AAVs. The termincludes the progeny of the original cell which has been transfected.Thus, a “host cell” as used herein may refer to a cell which has beentransfected with an exogenous DNA sequence. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

As used herein, the term “cell line” refers to a population of cellscapable of continuous or prolonged growth and division in vitro. Often,cell lines are clonal populations derived from a single progenitor cell.It is further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants.

As used herein, the terms “recombinant cell” refers to a cell into whichan exogenous DNA segment, such as DNA segment that leads to thetranscription of a biologically-active polypeptide or production of abiologically active nucleic acid such as an RNA, has been introduced.

As used herein, the term “vector” includes any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, artificial chromosome,virus, virion, etc., which is capable of replication when associatedwith the proper control elements and which can transfer gene sequencesbetween cells. Thus, the term includes cloning and expression vehicles,as well as viral vectors. In some embodiments, useful vectors arecontemplated to be those vectors in which the nucleic acid segment to betranscribed is positioned under the transcriptional control of apromoter. A “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. The phrases“operatively positioned,” “under control” or “under transcriptionalcontrol” means that the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene. The term “expression vector orconstruct” means any type of genetic construct containing a nucleic acidin which part or all of the nucleic acid encoding sequence is capable ofbeing transcribed. In some embodiments, expression includestranscription of the nucleic acid, for example, to generate abiologically--active polypeptide product or inhibitory RNA (e.g., shRNA,miRNA, miRNA inhibitor) from a transcribed gene.

The foregoing methods for packaging recombinant vectors in desired AAVcapsids to produce the rAAVs of the disclosure are not meant to belimiting and other suitable methods will be apparent to the skilledartisan.

Recombinant AAV Vectors

“Recombinant AAV (rAAV) vectors” of the disclosure are typicallycomposed of, at a minimum, a transgene and its regulatory sequences, and5′ and 3′ AAV inverted terminal repeats (ITRs). It is this recombinantAAV vector which is packaged into a capsid protein and delivered to aselected target cell. In some embodiments, the transgene is a nucleicacid sequence, heterologous to the vector sequences, which encodes apolypeptide, protein, functional RNA molecule (e.g., miRNA, miRNAinhibitor) or other gene product, of interest. The nucleic acid codingsequence is operatively linked to regulatory components in a mannerwhich permits transgene transcription, translation, and/or expression ina cell of a target tissue.

As used herein, a “target tissue” refers to a tissue of interest, e.g.,a tissue where it is desirable to deliver and/or express a transgene byAAV-mediated delivery. Target tissue may be healthy or diseased. Atarget tissue may be in vivo or ex vivo. In some embodiments, a targettissue contains one or more cell types present in an extracellularmatrix. For example, a target tissue may comprise tumor or cancer cellsand/or normal or non-transformed cells. A target tissue may be a tissueadversely affected by the improper function of a particular protein(e.g. non-functional ASPA in Canavan disease). In some embodiments,target tissue is blood, muscle, heart, pancreas and/or CNS tissue.

The AAV sequences of the vector typically comprise the cis-acting 5′ and3′ inverted terminal repeat sequences (See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168(1990)). The ITR sequences are about 145 bp in length. Preferably,substantially the entire sequences encoding the ITRs are used in themolecule, although some degree of minor modification of these sequencesis permissible. The ability to modify these ITR sequences is within theskill of the art. (See, e.g., texts such as Sambrook et al, “MolecularCloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory,New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). Anexample of such a molecule employed in the present disclosure is a“cis-acting” plasmid containing the transgene, in which the selectedtransgene sequence and associated regulatory elements are flanked by the5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained fromany known AAV, including presently identified may AAV types.

In addition to the major elements identified above for the recombinantAAV vector, the vector also includes conventional control elementsnecessary which are operably linked to the transgene in a manner whichpermits its transcription, translation and/or expression in a celltransfected with the plasmid vector or infected with the virus producedby the disclosure. As used herein, “operably linked” sequences includeboth expression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

As used herein, a nucleic acid sequence (e.g., coding sequence) andregulatory sequences are said to be “operably” linked when they arecovalently linked in such a way as to place the expression ortranscription of the nucleic acid sequence under the influence orcontrol of the regulatory sequences. If it is desired that the nucleicacid sequences be translated into a functional protein, two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably linked to a nucleic acidsequence if the promoter region were capable of effecting transcriptionof that DNA sequence such that the resulting transcript might betranslated into the desired protein or polypeptide. Similarly two ormore coding regions are operably linked when they are linked in such away that their transcription from a common promoter results in theexpression of two or more proteins having been translated in frame. Insome embodiments, operably linked coding sequences yield a fusionprotein. In some embodiments, operably linked coding sequences yield afunctional RNA (e.g., shRNA, miRNA, miRNA inhibitor).

For nucleic acids encoding proteins, a polyadenylation sequencegenerally is inserted following the transgene sequences and before the3′ AAV ITR sequence. A rAAV construct useful in the present disclosuremay also contain an intron, desirably located between thepromoter/enhancer sequence and the transgene. One possible intronsequence is derived from SV-40, and is referred to as the SV-40 T intronsequence. Another vector element that may be used is an internalribosome entry site (IRES). An IRES sequence is used to produce morethan one polypeptide from a single gene transcript. An IRES sequencewould be used to produce a protein that contain more than onepolypeptide chains. Selection of these and other common vector elementsare conventional and many such sequences are available [see, e.g.,Sambrook et al, and references cited therein at, for example, pages 3.183.26 and 16.1′7 16.27 and Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1989]. In some embodiments, a Footand Mouth Disease Virus 2A sequence is included in polyprotein; this isa small peptide (approximately 18 amino acids in length) that has beenshown to mediate the cleavage of polyproteins (Ryan, M D et al, EMBO,1994; 4: 928-933; Mattion, N Metal., J Virology, November 1996; p.8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin,C et al., The Plant Journal, 1999; 4: 453-459). The cleavage activity ofthe 2A sequence has previously been demonstrated in artificial systemsincluding plasmids and gene therapy vectors (AAV and retroviruses)(Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N Metal., JVirology, November 1996; p. 8124-8127; Furler, S et al., Gene Therapy,2001; 8: 864-873; and Halpin, C et al., The Plant Journal, 1999; 4:453-459; de Felipe, P et al., Gene Therapy, 1999; 6: 198-208; de Felipe,P et al., Human Gene Therapy, 2000; 11: 192.1-1931; and Klump, H et al.,Gene Therapy, 2001; 8: 811-817).

The precise nature of the regulatory sequences needed for geneexpression in host cells may vary between species, tissues or celltypes, but shall in general include, as necessary, 5′ non-transcribedand 5′ non-translated sequences involved with the initiation oftranscription and translation respectively, such as a TATA box, cappingsequence, CAAT sequence, enhancer elements, and the like. Especially,such 5′ non-transcribed regulatory sequences will include a promoterregion that includes a promoter sequence for transcriptional control ofthe operably joined gene. Regulatory sequences may also include enhancersequences or upstream activator sequences as desired. The vectors of thedisclosure may optionally include 5′ leader or signal sequences. Thechoice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1αpromoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); theecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA,93:3346-3351 (1996)), the tetracycline-repressible system (Gossen et al,Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), thetetracycline-inducible system (Gossen et al, Science, 268:1766-1769(1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518(1998)), the RU486-inducible system (Wang et al, Nat. Biotech.,15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)) and therapamycin-inducible system (Magari et al, J. Clin. Invest.,100:2865-2872 (1997)). Still other types of inducible promoters whichmay be useful in this context are those which are regulated by aspecific physiological state, e.g., temperature, acute phase, aparticular differentiation state of the cell, or in replicating cellsonly.

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

In some embodiments, the regulatory sequences impart tissue-specificgene expression capabilities. In some cases, the tissue-specificregulatory sequences bind tissue-specific transcription factors thatinduce transcription in a tissue specific manner. Such tissue-specificregulatory sequences promoters, enhancers, etc.) are well known in theart. Exemplary tissue-specific regulatory sequences include, but are notlimited to the following tissue specific promoters: a liver-specificthyroxin binding globulin (TBG) promoter, a insulin promoter, a glucagonpromoter, a somatostatin promoter, a pancreatic polypeptide (PPY)promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter,a mammalian desmin (DES) promoter, a α-myosin heavy chain (a-MHC)promoter, or a cardiac Troponin (cTnT) promoter. Other exemplarypromoters include Beta-actin promoter, hepatitis B virus core promoter,Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP)promoter, Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), boneosteocalcin promoter (Stein et al, Mol. Biol. Rep., 24:185-96 (1997));bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64(1996)), CD2 promoter (, Hansal et al., J. Immunol, 161:1063-8 (1998);immunoalobulin heavy chain promoter; T cell receptor α-chain promoter,neuronal such as neuron-specific enolase (NSE) promoter (Andersen etal., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light-chaingene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5(1991)), and the neuron-specific vgf gene promoter (Piccioli et al.,Neuron, 15:373-84 (1995)), among others which will be apparent to to theskilled artisan.

In some embodiments, one or more bindings sites for one or more ofmiRNAs are incorporated in a transgene of a rAAV vector, to inhibit theexpression of the transgene in one or more tissues of an subjectharboring the transgene. The skilled artisan will appreciate thatbinding sites may be selected to control the expression of a trangene ina tissue specific manner. For example, binding sites for theliver-specific miR-122 may be incorporated into a transgene to inhibitexpression of that transgene in the liver. The target sites in the mRNAmay be in the 5′ UTR, the 3′ UTR or in the coding region. Typically, thetarget site is in the 3′ UTR of the mRNA. Furthermore, the transgene maybe designed such that multiple miRNAs regulate the mRNA by recognizingthe same or multiple sites. The presence of multiple miRNA binding sitesmay result in the cooperative action of multiple RISCs and providehighly efficient inhibition of expression. The target site sequence maycomprise a total of 5-100, 10-60, or more nucleotides. The target sitesequence may comprise at least 5 nucleotides of the sequence of a targetgene binding site.

Recombinant AAV Vector: Transgene Coding Sequences

The composition of the transgene sequence of the rAAV vector will dependupon the use to which the resulting vector will be put. For example, onetype of transgene sequence includes a reporter sequence, which uponexpression produces a detectable signal. In another example, thetrangene encodes a therapeutic protein or therapeutic functional RNA. Inanother example, the transgene encodes a protein or functional RNA thatis intended to be used for research purposes, e.g., to create a somatictransgenic animal model harboring the transgene, e.g., to study thefunction of the transgene product. In another example, the transgeneencodes a protein or functional RNA that is intended to be used tocreate an animal model of disease. Appropriate transgene codingsequences will be apparent to the skilled artisan.

Reporter sequences that may be provided in a transgene include, withoutlimitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, and others wellknown in the art. When associated with regulatory elements which drivetheir expression, the reporter sequences, provide signals detectable byconventional means, including enzymatic, radiographic, colorimetric,fluorescence or other spectrographic assays, fluorescent activating cellsorting assays and immunological assays, including enzyme linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) andimmunohistochemistry. For example, where the marker sequence is the LacZgene, the presence of the vector carrying the signal is detected byassays for β-galactosidase activity. Where the transgene is greenfluorescent protein or luciferase, the vector carrying the signal may bemeasured visually by color or light production in a luminometer. Suchreporters can, for example, be useful in verifying the tissue-specifictargeting capabilities and tissue specific promoter regulatory activityof an rAAV.

In some aspects, the disclosure provides rAAV vectors for use in methodsof preventing or treating one or more genetic deficiencies ordysfunctions in a mammal, such as for example, a polypeptide deficiencyor polypeptide excess in a mammal, and particularly for treating orreducing the severity or extent of deficiency in a human manifesting oneor more of the disorders linked to a deficiency in such polypeptides incells and tissues. The method involves administration of an rAAV vectorthat encodes one or more therapeutic peptides, polypeptides, siRNAs,microRNAs, antisense nucleotides, etc. in a pharmaceutically-acceptablecarrier to the subject in an amount and for a period of time. sufficientto treat the deficiency or disorder in the subject suffering from such adisorder.

Thus, the disclosure embraces the delivery of rAAV vectors encoding oneor more peptides, polypeptides, or proteins, which are useful for thetreatment or prevention of disease states in a mammalian subject.Exemplary therapeutic proteins include one or more polypeptides selectedfrom the group consisting of growth factors, interleukins, interferons,anti-apoptosis factors, cytokines, anti-diabetic factors, anti-apoptosisagents, coagulation factors, anti-tumor factors. Other non-limitingexamples of therapeutic proteins include BDNF, CNTF, CSF, EGF, FGE,G-SCF, GM-CSF, gonadotropin, IFN, IFG-1, M-CSE, NGF, PDGF, PEDF, TGP,VEGF, TGF-B2, TNF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(187A), viral IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16 IL-17, and IL-18.

The rAAV vectors may comprise a gene to be transferred to a subject totreat a disease associated with reduced expression, lack of expressionor dysfunction of the gene. In some embodiments, the dysfunctional geneis ASPA and the disease is Canavan disease.

Exemplary genes and associated disease states include, but are notlimited to: glucose-6-phosphatase, associated with glycogen storagedeficiency type 1A; phosphoenolpyruvate-carboxykinases, associated withPepck deficiency; galactose-1 phosphate uridyl transferase, associatedwith galactosemia; phenylalanine hydroxylase, associated withphenylketonuria; branched chain alpha-ketoacid dehydrogenase, associatedwith Maple syrup urine disease; fumarylacetoacetate hydrolase,associated with tyrosinemia type 1; methylmalonyl-CoA mutase, associatedwith methylmalonic acidemia; medium chain acyl CoA dehydrogenase,associated with medium chain acetyl CoA deficiency; omithinetranscarbamylase, associated with omithine transcarbamylase deficiency;argininosuccinic acid synthetase, associated with citrullinemia; lowdensity lipoprotein receptor protein, associated with familialhypercholesterolemia; UDP-glucouronosyltransferase, associated withCrigler-Najjar disease; adenosine deaminase, associated with severecombined immunodeficiency disease; hypoxanthine guanine phosphoribosyltransferase, associated with Gout and Lesch-Nyan syndrome; biotinidase,associated with biotinidase deficiency; beta-glucocerebrosidase,associated with Gaucher disease; beta-glucuronidase, associated with Slysyndrome; peroxisome membrane protein 70 kDa, associated with Zellwegersyndrome; porphobilinogen deaminase, associated with acute intermittentporphyria; alpha-1 antitrypsin for treatment of alpha-1 antitrypsindeficiency (emphysema); erythropoietin for treatment of anemia due tothalassemia or to renal failure; vascular endothelial growth factor,angiopoietin-1, and fibroblast growth factor for the treatment ofischemic diseases; thrombomodulin and tissue factor pathway inhibitorfor the treatment of occluded blood vessels as seen in, for example,atherosclerosis, thrombosis, or embolisms; aromatic amino aciddecarboxylase (AADC), and tyrosine hydroxylase (TH) for the treatment ofParkinson's disease; the beta adrenergic receptor, anti-sense to, or amutant form of, phospholamban, the sarco(endo)plasmic reticulumadenosine triphosphatase-2 (SERCA2), and the cardiac adenylyl cyclasefor the treatment of congestive heart failure; a tumor suppessor genesuch as p53 for the treatment of various cancers; a cytokine such as oneof the various interleukins for the treatment of inflammatory and immunedisorders and cancers; dystrophin or minidystrophin and utrophin orminiutrophin for the treatment of muscular dystrophies; and, insulin forthe treatment of diabetes.

In some embodiments, The rAAV vectors may comprise a gene encoding anantigen-binding protein, such as an immunoglobulin heavy chain or lightchain or fragment thereof, e.g., that may be used for therapeuticpurposes. In some embodiments, the protein is a single chain Fv fragmentor Fv-Pc fragment. Accordingly, in some embodiments, the rAAV can beused to infect cells of a target tissue (e.g., muscle tissue) so as toengineer cells of the tissue to express an antigen-binding protein, suchas an antibody or fragment thereof. In some embodiments, to generaterAAVs that express the antibodies or antigen binding fragments, cDNAsengineered to express such proteins will be sucloned into an appropriateplasmid backbone and packaged into an rAAV.

The rAAVs of the disclosure can be used to restore the expression ofgenes that are reduced in expression, silenced, or otherwisedysfunctional in a subject (e.g., a tumor suppressor that has beensilenced in a subject having cancer). The rAAVs of the disclosure canalso be used to knockdown the expression of genes that are aberrantlyexpressed in a subject (e.g., an oncogene that is expressed in a subjecthaving cancer). In some embodiments, an rAAV vector comprising a nucleicacid encoding a gene product associated with cancer (e.g., tumorsuppressors) may be used to treat the cancer, by administering a rAAVharboring the rAAV vector to a subject having the cancer. In someembodiments, an rAAV vector comprising a nucleic acid encoding a smallinterfering nucleic acid (e.g., shRNAs, miRNAs) that inhibits theexpression of a gene product associated with cancer (e.g., oncogenes)may be used to treat the cancer, by administering a rAAV harboring therAAV vector to a subject having the cancer. In some embodiments, an rAAVvector comprising a nucleic acid encoding a gene product associated withcancer (or a functional RNA that inhibits the expression of a geneassociated with cancer) may be used for research purposes, e.g., tostudy the cancer or to identify therapeutics that treat the cancer. Thefollowing is a non-limiting list of exemplary genes known to beassociated with the development of cancer (e.g., oncogenes and tumorsuppressors): AARS, ABCB1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1,ADSL, AK1, AKR1C2, AKT1, ALB, ANPEP, ANXA5, ANXA7, AP2M1, APC, ARHGAP5,ARHGEF5, ARID4A, ASNS, ATF4, ATM, ATP5B, ATP5O, AXL, BARD1, BAX, BCL2,BHLHB2, BLMH, BRAF, BRCA1, BRCA2, BTK, CANX, CAP1, CAPN1, CAPNS1, CAV1,CBFB, CBLB, CCL2, CCND1, CCND2, CCND3, CCNE1, CCT5, CCYR61, CD24, CD44,CD59, CDC20, CDC25, CDC25A, CDC25B, CDC2L5, CDK10, CDK4, CDK5, CDK9,CDKL1, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG, CENPC1,CGRRF1, CHAF1A, CIB1, CKMT1, CLK1, CLK2, CLK3, CLNS1A, CLTC, COL1A1,COL6A3, COX6C, COX7A2, CRAT, CRHR1, CSF1R, CSK, CSNK1G2, CTNNA1, CTNNB1,CTPS, CTSC, CTSD, CUL1, CYR61, DCC, DCN, DDX10, DEK, DHCR7, DHRS2, DHX8,DLG3, DVL1, DVL3, E2F1, E2F3, E2F5, EGFR, EGR1, EIF5, EPHA2, ERBB2,ERBB3, ERBB4, ERCC3, ETV1, ETV3, ETV6, F2R, FASTK, FBN1, FBN2, FES,FGFR1, FGR, FKBP8, FN1, FOS, FOSL1, FOSL2, FOXG1A, FOXO1A, FRAP1, FRZB,FTL, FZD2, FZD5, FZD9, G22P1, GAS6, GCN5L2, GDF15, GNA13, GNAS, GNB2,GNB2L1, GPR39, GRB2, GSK3A, GSPT1, GTF2I, HDAC1, HDGF, HMMR, HPRT1, HRB,HSPA4, HSPA5, HSPA8, HSPB1, HSPH1, HYAL1, HYOU1, ICAM1, ID1, ID2, IDUA,IER3, IGF1R, IGF2R, IGFBP3, IGFBP4, IGFBP5, IL1B, ILK, ING1, IRF3,ITGA3, ITGA6, ITGB4, JAK1, JARID1A, JUN, JUNB, JUND, K-ALPHA-1, KIT,KITLG, KLK10, KPNA2, KRAS2, KRT18, KRT2A, KRT9, LAMB1, LAMP2, LCK, LCN2,LEP, LITAF, LRPAP1, LTF, LYN, LZTR1, MADH1, MAP2K2, MAP3K8, MAPK12,MAPK13, MAPKAPK3, MAPRE1, MARS, MAS1, MCC, MCM2, MCM4, MDM2, MDM4, MET,MGST1, MICB, MLLT3, MME, MMP1, MMP14, MMP17, MMP2, MNDA, MSH2, MSH6,MT3, MYB, MYBL1, MYBL2, MYC, MYCL1, MYCN, MYD88, MYL9, MYLK, NEO1, NF1,NF2, NFKB1, NFKB2, NFSF7, NID, NINJ1, NMBR, NME1, NME2, NME3, NOTCH1,NOTCH2, NOTCH4, NPM1, NQO1, NR1D1, NR2F1, NR2F6, NRAS, NRG1, NSEP1, OSM,PA2G4, PABPC1, PCNA, PCTK1, PCTK2, PCTK3, PDGFA, PDGFB, PDGRA, PDPK1,PEA15, PFDN4, PFDN5, PGAM1, PHB, PIK3CA, PIK3CB, PIK3CG, PIM1, PKM2,PKMYT1, PLK2, PPARD, PPARG, PPIH, PPP1CA, PPP2R5A, PRDX2, PRDX4,PRKAR1A, PRKCBP1, PRNP, PRSS15, PSMA1, PTCH, PTEN, PTGS1, PTMA, PTN,PTPRN, RAB5A, RAC1, RAD50, RAF1, RALBP1, RAP1A, RARA, RARB, RASGRF1,RB1, RBBP4, RBL2, REA, REL, RELA, RELB, RET, RFC2, RGS19, RHOA, RHOB,RHOC, RHOD, RIPK1, RPN2, RPS6KB1, RRM1, SARS, SELENBP1, SEMA3C, SEMA4D,SEPP1, SERPINH1, SFN, SFPQ, SFRS7, SHB, SHH, SIAH2, SIVA, SIVA TP53,SKI, SKIL, SLC16A1, SLC1A4, SLC20A1, SMO, SMPD1, SNAI2, SND1, SNRPB2,SOCS1, SOCS3, SOD1, SORT1, SPINT2, SPRY2, SRC, SRPX, STAT1, STAT2,STAT3, STAT5B, STC1, TAF1, TBL3, TBRG4, TCF1, TCF7L2, TFAP2C, TFDP1 ,TFDP2, TGFA, TGFB1, TGFB1, TGFBR2, TGFBR3, THBS1, TIE, TIMP1, TIMP3,TJP1, TK1, TLE1, TNF, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B, TNERSF6,TNFSF7, TNK1, TOB1, TP53, TP53BP2, TP53I3, TP73, TPBG, TPT1, TRADD,TRAM1, TRRAP, TSG101, TUFM, TXNRD1, TYRO3, UBC, UBE2L6, UCHL1, USP7,VDAC1, VEGF, VHL, VIL2, WEE1, WNT1, WNT2, WNT2B, WNT3, WNT5A, WT1,XRCC1, YES 1, YWHAB, YWHAZ, ZAP70, and ZNF9.

In some embodiments, rAAV vectors comprising a transgene encoding atumor suppressor protein are useful for the treatment of cancer. As usedherein, the term “tumor suppressor protein” refers to protein thatinhibits the formation, development or maintenance of a tumor. In someembodiments, a tumor suppressor protein suppresses regulation of thecell cycle and/or promotes apoptosis. Loss or dysfunction of tumorsuppressor proteins is a key step in the development of cancer.Accordingly, expression of tumor suppressor proteins inhibits or slowsthe growth of cancerous tumors and may therefore be a viable therapeuticstrategy for treatment of cancer. In some embodiments, the tumorsuppressor protein is a secreted tumor suppressor protein. Non-limitingexamples of secreted tumor suppressor proteins include IGEBP7, SRPX, andothers disclosed for example in Min Zhao, Jingchun Sun, Zhongming Zhao(2013) TSGene: a web resource for tumor suppressor genes. Nucleic AcidsResearch, 41: D970-D976.

A rAAV vector may comprise as a transgene, a nucleic acid encoding aprotein or functional RNA that modulates apoptosis. The following is anon-limiting list of genes associated with apoptosis and nucleic acidsencoding the products of these genes and their homologues and encodingsmall interfering nucleic acids (e.g., shRNAs, miRNAs) that inhibit theexpression of these genes and their homologues are useful as transgenesin certain embodiments of the disclosure: RPS27A, ABL1, AKT1, APAF1,BAD, BAG1, BAG3, BAG4, BAK1, BAX, BCL10, BCL2, BCL2A1, BCL2L1, BCL2L10,BCL2L11, BCL2L12, BCL2L13, BCL2L2, BCLAF1, BFAR, BID, BIK, NAIP, BIRC2,BIRC3, XIAP, BIRC5, BIRC6, BIRC7, BIRC8, BNIP1, BNIP2, BNIP3, BNIP3L,BOK, BRAF, CARD10, CARD11, NLRC4, CARD14, NOD2, NOD1, CARD6, CARD8,CARD9, CASP1, CASP10, CASP14, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7,CASP8, CASP9, CFLAR, CIDEA, CIDEB, CRADD, DAPK1, DAPK2, DFFA, DFFB,FADD, GADD45A, GDNF, HRK, IGF1R, LTA, LTBR, MCL1, NOL3, PYCARD, RIPK1,RIP2, TNF, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B,TNFRSF12A, TNFRSF14, TNFRSF19, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF25,CD40, FAS, TNFRSF6B, CD27, TNFRSF9, TNFSF10, TNFSF14, TNFSF18, CD40LG,FASLG, CD70, TNFSF8, TNFSF9, TP53, TP53BP2, TP73, TP63, TRADD, TRAF1,TRAF2, TRAF3, TRAF4, and TRAF5.

The skilled artisan will also realize that in the case of transgenesencoding proteins or polypeptides, that mutations that results inconservative amino acid substitutions may be made in a transgene toprovide functionally equivalent variants, or homologs of a protein orpolypeptide. In some aspects the disclosure embraces sequencealterations that result in conservative amino acid substitution of atransgene. In some embodiments, the transgene comprises a gene having adominant negative mutation. For example, a transgene may express amutant protein that interacts with the same elements as a wild-typeprotein, and thereby blocks some aspect of the function of the wild-typeprotein.

Useful transgene products also include miRNAs. miRNAs and other smallinterfering nucleic acids regulate gene expression via target RNAtranscript cleavage/degradation or translational repression of thetarget messenger RNA (mRNA). miRNAs are natively expressed, typically asfinal 19-25 non-translated RNA products. miRNAs exhibit their activitythrough sequence-specific interactions with the 3′ untranslated regions(UTR) of target mRNAs. These endogenously expressed miRNAs form hairpinprecursors which are subsequently processed into a miRNA duplex, andfurther into a “mature” single stranded miRNA molecule. This maturemiRNA guides a multiprotein complex, miRISC, which identifies targetsite, e.g., in the 3′ UTR regions, of target mRNAs based upon theircomplementarity to the mature miRNA.

The following non-limiting list of miRNA genes, and their homologues,are useful as transgenes or as targets for small interfering nucleicacids encoded by transgenes (e.g., miRNA sponges, antisenseoligonucleotides, TuD RNAs) in certain embodiments of the methods:hsa-let-7a, hsa-let-7a*, hsa-let-7b, hsa-let-7b*, hsa-let-7c,hsa-let-7c*, hsa-let-7d, hsa-let-7d*, hsa-let-7e, hsa-let-7e*,hsa-let-7f, hsa-let-7f-1*, hsa-let-7f-2*, hsa-let-7g, hsa-let-7g*,hsa-let-7i, hsa-let-7i*, hsa-miR-1, hsa-miR-100, hsa-miR-100*,hsa-miR-101, hsa-miR-101*, hsa-miR-103, hsa-miR 105, hsa-miR-105*,hsa-miR-106a, hsa-miR-106a*, has-miR-106b, hsa-miR-106b*, hsa-miR-107,hsa-miR-10a, hsa-miR-10a*, hsa-miR-10b, hsa-miR-10b*, hsa-miR-1178,hsa-miR-1179, hsa-miR-1180, hsa-miR-1181, hsa-miR-1182, hsa-miR-1183,hsa-miR-1184, hsa-miR-1185, hsa-miR-1197, hsa-miR-1200, hsa-miR-1201,hsa-miR-1202, hsa-miR-1203, hsa-miR-1204, hsa-miR-1205, hsa-miR-1206,hsa-miR-1207-3p, hsa-miR-1207-5p, hsa-miR-1208, hsa-miR-122,hsa-miR-122*, hsa-miR-1224-3p, hsa-miR-1224-5p, has-miR-1225-3p,hsa-miR-1225-5p, hsa-miR-1226, hsa-miR-1226*, hsa-miR-1227,hsa-miR-1228, hsa-miR-1228*, hsa-miR-1229, hsa-miR-1231, hsa-miR-1233,hsa-miR-1234, hsa-miR-1236, hsa-miR-1237, hsa-miR-1238, hsa-miR-124,hsa-miR-124*, hsa-miR-1243, hsa-miR-1244, hsa-miR-1245, hsa-miR-1246,hsa-miR-1247, hsa-miR-1248, hsa-miR-1249, hsa-miR-1250, hsa-miR-1251,hsa-miR-1252, hsa-miR-1253, hsa-miR-1254, hsa-miR-1255a, hsa-miR-1255b,hsa-miR-1256, hsa-miR-1257, hsa-miR-1258, hsa-miR-1259, hsa-miR-125a-3p,hsa-miR-125a-5p, hsa-miR-125b, hsa-miR-125b-1*, hsa-miR-125b-2*,hsa-miR-126, hsa-miR-126*, hsa-miR-1260, hsa-miR-1261, hsa-miR-1262,hsa-miR-1263, hsa-miR-1264, hsa-miR-1265, hsa-miR-1266, hsa-miR-1267,hsa-miR-1268, hsa-miR-1269, hsa-miR-1270, hsa-miR-1271, hsa-miR-1272,hsa-miR-1273, hsa-miR-127--3p, hsa-miR-1274a, hsa-miR-1274b,hsa-miR-1275, hsa-miR-127-5p, hsa-miR-1276, hsa-miR-1277, hsa-miR-1278,hsa-miR-1279, hsa-miR-128, hsa-miR-1280, hsa-miR-1281, hsa-miR-1282,hsa-miR-1283, hsa-miR-1284, hsa-miR-1285, hsa-miR-1286, hsa-miR-1287,hsa-miR-1288, hsa-miR-1289, hsa-miR-129*, hsa-miR-1290, hsa-miR-1291,hsa-miR-1292, hsa-miR-1293, hsa-miR-129-3p, has-miR-1294, hsa-miR-1295,hsa-miR-129-5p, hsa-miR-1296, hsa-miR-1297, hsa-miR-1298, hsa-miR-1299,hsa-miR-1300, hsa-miR-1301, hsa-miR-1302, hsa-miR-1303, hsa-miR-1304,hsa-miR-1305, hsa-miR-1306, hsa-miR-1307, hsa-miR-1308, hsa-miR-130a,hsa-miR-130a*, hsa-miR-130b, hsa-miR-130b*, hsa-miR-132, hsa-miR-132*,hsa-miR-1321, hsa-miR-1322, hsa-miR-1323, hsa-miR-1324, hsa-miR-133a,hsa-miR-133b, hsa-miR-134, hsa-miR-135a, hsa-miR-135a*, hsa-miR-135b,hsa-miR-135b*, hsa-miR-136, hsa-miR-136*, hsa-miR-137, hsa-miR-138,hsa-miR-138-1*, hsa-miR-138-2*, hsa-miR-139-3p, hsa-miR-139-5p,hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141, hsa-miR-141*,hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-143, hsa-miR-143*, has-miR-144,hsa-miR-144*, hsa-miR-145, hsa-miR-145*, hsa-miR-146a, hsa-miR-146a*,hsa-miR-146b-3p, hsa-miR-146b-5p, hsa-miR-147, hsa-miR-147b,hsa-miR-148a, hsa-miR-148a*, hsa-miR-148b, hsa-miR-148b*, hsa-miR-149,hsa-miR-149*, hsa-miR-150, hsa-miR-150*, hsa-miR-151-3p, hsa-miR-151-5p,hsa-miR-152, hsa-miR-153, hsa-miR-154, hsa-miR-154*, hsa-miR-155,hsa-miR-155*, hsa-miR-15a, hsa-miR-15a*, hsa-miR-15b, hsa-miR-15b*,hsa-miR-16, hsa-miR-16-1*, hsa-miR-16-2*, hsa-miR-17, hsa-miR-17*,hsa-miR-181a, hsa-miR-181a*, hsa-miR-181a-2*, hsa-miR-181b,hsa-miR-181c, hsa-miR-181c*, hsa-miR-181d, hsa-miR-182, hsa-miR-182*,hsa-miR-1825, hsa-miR-1826, hsa-miR-1827, hsa-miR-183, hsa-miR-183*,hsa-miR-184, hsa-miR-185, hsa-miR-185*, hsa-miR-186, hsa-miR-186*,hsa-miR-187, hsa-miR-187*, hsa-miR-188-3p, hsa-miR-188-5p, hsa-miR-18a,hsa-miR-18a*, hsa-miR-18b, hsa-miR-18b*, hsa-miR-190, hsa-miR-190b,hsa-miR-191, hsa-miR-191*, hsa-miR-192, hsa-miR-192*, hsa-miR-193a-3p,hsa-miR-193a-5p, hsa-miR-193b, hsa-miR-193b*, hsa-miR-194, hsa-miR-194*,hsa-miR-195, hsa-miR-195*, hsa-miR-196a, hsa-miR-196a*, hsa-miR-196b,hsa-miR-197, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-199a-5p,hsa-miR-199b-5p, hsa-miR-19a, hsa-miR-19a*, hsa-miR-19b, hsa-miR-19b-1,hsa-miR-19b-2*, hsa-miR-200a, hsa-miR-200a*, hsa-miR-200b,hsa-miR-200b*, hsa-miR-200c, hsa-miR-200c*, hsa-miR-202, hsa-miR-202*,hsa-miR-203, hsa-miR-204, hsa-miR-205, hsa-miR-206, hsa-miR-208a,hsa-miR-208b, hsa-miR-20a, hsa-miR-20a*, hsa-miR-20b, hsa-miR-20b*,hsa-miR-21, hsa-miR-21*, hsa-miR-210, hsa-miR-211, hsa-miR-212,hsa-miR-214, hsa-miR-214*, hsa-miR-215, hsa-miR-216a, hsa-miR-216b,hsa-miR-217, hsa-miR-218, hsa-miR-218-1*, hsa-miR-218-2*,hsa-miR-219-1-3p, hsa-miR-219-2-3p, hsa-miR-219-5p, hsa-miR-22,hsa-miR-22*, hsa-miR-220a, hsa-miR-220b, hsa-miR-220c, hsa-miR-221,hsa-miR-221*, hsa-miR-222, hsa-miR-222*, hsa-miR-223, hsa-miR-223*,hsa-miR-224, hsa-miR-23a, hsa-miR-23a*, hsa-miR-23b, hsa-miR-23b*,hsa-miR-24, hsa-miR-24-1*, hsa-miR-24-2*, hsa-miR-25, hsa-miR-25*,hsa-miR-26a, hsa-miR-26a-1*, hsa-miR-26a-2*, hsa-miR-26b, hsa-miR-26b*,has-miR-27a, hsa-miR-27a*, hsa-miR-27b, hsa-miR-27b*, hsa-miR-28-3p,hsa-miR-28-5p, hsa-miR-296-3p, hsa-miR-296-5p, hsa-miR-297, hsa-miR-298,hsa-miR-299-3p, hsa-miR-299-5p, hsa-miR-29a, hsa-miR-29a*, hsa-miR-29b,hsa-miR-29b-1*, hsa-miR-29b-2*, hsa-miR-29c, hsa-miR-29c*, hsa-miR-300,hsa-miR-301a, hsa-miR-301b, hsa-miR-302a, hsa-miR-302a*, hsa-miR-302b,hsa-miR-302b*, hsa-miR-302c, hsa-miR-302c*, hsa-miR-302d, hsa-miR-302d*,hsa-miR-302e, hsa-miR-302f, hsa-miR-30a, hsa-miR-30a*, hsa-miR-30b,hsa-miR-30b*, hsa-miR-30c, hsa-miR-30c-1*, hsa-miR-30c-2*, hsa-miR-30d,hsa-miR-30d*, hsa-miR-30e, hsa-miR-30e*, hsa-miR-31, hsa-miR-31*,hsa-miR-32, hsa-miR-32*, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c,hsa-miR-320d, hsa-miR-323-3p, hsa-miR-323-5p, hsa-miR-324-3p,hsa-miR-324-5p, hsa-miR-325, hsa-miR-326, hsa-miR-329, hsa-miR-330-3p,hsa-miR-330-5p, hsa-miR-331-3p, hsa-miR-331-5p, hsa-miR-335,hsa-miR-335*, hsa-miR-337-3p, hsa-miR-337-5p, hsa-miR-338-3p,hsa-miR-338-5p, hsa-miR-339-3p, hsa-miR-339-5p, hsa-miR-33a,hsa-miR-33a*, hsa-miR-33b, hsa-miR-33b*, hsa-miR-340, hsa-miR-340*,hsa-miR-342-3p, hsa-miR-342-5p, hsa-miR-345, hsa-miR-346, hsa-miR-34a,hsa-miR-34a*, hsa-miR-34b, hsa-miR-34b*, hsa-miR-34c-3p, hsa-miR-34c-5p,hsa-miR-361-3p, hsa-miR-361-5p, hsa-miR-362-3p, hsa-miR-362-5p,hsa-miR-363, hsa-miR-363*, hsa-miR-365, hsa-miR-367, hsa-miR-367*,hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-370, hsa-miR-371-3p,hsa-miR-371-5p, hsa-miR-372, hsa-miR-373, hsa-miR-373*, hsa-miR-374a,hsa-miR-374a*, hsa-miR-374b, hsa-miR-374b*, hsa-miR-375, hsa-miR-376a,hsa-miR-376a*, hsa-miR-376b, hsa-miR-376c, hsa-miR-377, hsa-miR-377*,hsa-miR-378, hsa-miR-378*, hsa-miR-379, hsa-miR-379*, hsa-miR-380,hsa-miR-380*, hsa-miR-381, hsa-miR-382, hsa-miR-383, hsa-miR-384,hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410, hsa-miR-411, hsa-miR-411*,hsa-miR-412, hsa-miR-421, hsa-miR-422a, hsa-miR-423-3p, hsa-miR-423-5p,hsa-miR-424, hsa-miR-424*, hsa-miR-425, hsa-miR-425*, hsa-miR-429,hsa-miR-431, hsa-miR-431*, hsa-miR-432, hsa-miR-432*, hsa-miR-433,hsa-miR-448, hsa-miR-449a, hsa-miR-449b, hsa-miR-450a, hsa-miR-450b-3p,hsa-miR-450b-5p, hsa-miR-451, hsa-miR-452, hsa-miR-452*, hsa-miR-453,hsa-miR-454, hsa-miR-454*, hsa-miR-455-3p, hsa-miR-455-5p,hsa-miR-483-3p, hsa-miR-483-5p, hsa-miR-484, hsa-miR-485-3p,hsa-miR-485-5p, hsa-miR-486-3p, hsa-miR-486-5p, hsa-miR-487a,hsa-miR-487b, hsa-miR-488, hsa-miR-488*, hsa-miR-489, hsa-miR-490-3p,hsa-miR-490-5p, hsa-miR-491-3p, hsa-miR-491-5p, hsa-miR-492,hsa-miR-493, hsa-miR-493*, hsa-miR-494, hsa-miR-495, hsa-miR-496,hsa-miR-497, hsa-miR-497*, hsa-miR-498, hsa-miR-499-3p, hsa-miR-499-5p,hsa-miR-500, hsa-miR-500*, hsa-miR-501-3p, hsa-miR-501-5p,hsa-miR-502-3p, hsa-miR-502-5p, hsa-miR-503, hsa-miR-504, hsa-miR-505,hsa-miR-505*, hsa-miR-506, hsa-miR-507, hsa-miR-508-3p, hsa-miR-508-5p,hsa-miR-509-3-5p, hsa-miR-509-3p, hsa-miR-509-5p, hsa-miR-510,hsa-miR-511, hsa-miR-512-3p, hsa-miR-512-5p, hsa-miR-513a-3p,hsa-miR-513a-5p, hsa-miR-513b, hsa-miR-513c, hsa-miR-514,hsa-miR-515-3p, hsa-miR-515-5p, hsa-miR-516a-3p, hsa-miR-516a-5p,hsa-miR-516b, hsa-miR-517*, hsa-miR-517a, hsa-miR-517b, hsa-miR-517c,hsa-miR-518a-3p, hsa-miR-518a-5p, hsa-miR-518b, hsa-miR-518c,hsa-miR-518c*, hsa-miR-518d-3p, hsa-miR-518d-5p, hsa-miR-518e,hsa-miR-518e*, hsa-miR-518f, hsa-miR-518f, hsa-miR-519a,hsa-miR-519b-3p, hsa-miR-519c-3p, hsa-miR-519d, hsa-miR-519e,hsa-miR-519e*, hsa-miR-520a-3p, hsa-miR-520a-5p, hsa-miR-520b,hsa-miR-520c-3p, hsa-miR-520d-3p, hsa-miR-520d-5p, hsa-miR-520e,hsa-miR-520f, hsa-miR-520g, hsa-miR-520h, hsa-miR-521, hsa-miR-522,hsa-miR-523, hsa-miR-524-3p, hsa-miR-524-5p, hsa-miR-525-3p,hsa-miR-525-5p, hsa-miR-526b, hsa-miR-526b*, hsa-miR-532-3p,hsa-miR-532-5p, hsa-miR-539, hsa-miR-541, hsa-miR-541*, hsa-miR-542-3p,hsa-miR-542-5p, hsa-miR-543, hsa-miR-544, hsa-miR-545, hsa-miR-545*,hsa-miR-548a-3p, hsa-miR-548a-5p, hsa-miR-540-3p, hsa-miR-548b-5p,hsa-miR-548c-3p, hsa-miR-548c-5p, hsa-miR-548d-3p, hsa-miR-548d-5p,hsa-miR-548e, hsa-miR-548f, hsa-miR-548g, hsa-miR-548h, hsa-miR-548i,hsa-miR-548j, hsa-miR-548k, hsa-miR-548l, hsa-miR-548m, hsa-miR-548n,hsa-miR-548o, hsa-miR-548p, hsa-miR-549, hsa-miR-550, hsa-miR-550*,hsa-miR-551a, hsa-miR-551b, hsa-miR-551b*, hsa-miR-552, hsa-miR-553,hsa-miR-554, hsa-miR-555, hsa-miR-556-3p, hsa-miR-556-5p, hsa-miR-557,to hsa-miR-558, hsa-miR-559, hsa-miR-561, hsa-miR-562, hsa-miR-563,hsa-miR-564, hsa-miR-566, hsa-miR-567, hsa-miR-568, hsa-miR-569,hsa-miR-570, hsa-miR-571, hsa-miR-572, hsa-miR-573, hsa-miR-574-3p,hsa-miR-574-5p, hsa-miR-575, hsa-miR-576-3p, hsa-miR-576-5p,hsa-miR-577, hsa-miR-578, hsa-miR-579, hsa-miR-580, hsa-miR-581,hsa-miR-582-3p, hsa-miR-582-5p, hsa-miR-583, hsa-miR-584, hsa-miR-585,hsa-miR-586, hsa-miR-587, hsa-miR-588, hsa-miR-589, hsa-miR-589*,hsa-miR-590-3p, hsa-miR-590-5p, hsa-miR-591, hsa-miR-592, hsa-miR-593,hsa-miR-593*, hsa-miR-595, hsa-miR-596, hsa-miR-597, hsa-miR-598,hsa-miR-599, hsa-miR-600, hsa-miR-601, hsa-miR-602, hsa-miR-603,hsa-miR-604, hsa-miR-605, hsa-miR-606, hsa-miR-607, hsa-miR-608,hsa-miR-609, hsa-miR-610, hsa-miR-611, hsa-miR-612, hsa-miR-613,hsa-miR-614, hsa-miR-615-3p, hsa-miR-615-5p, hsa-miR-616, hsa-miR-616*,hsa-miR-617, hsa-miR-618, hsa-miR-619, hsa-miR-620, hsa-miR-621,hsa-miR-622, hsa-miR-623, hsa-miR-624, hsa-miR-624*, hsa-miR-625,hsa-miR-625*, hsa-miR-626, hsa-miR-627, hsa-miR-628-3p, hsa-miR-628-5p,hsa-miR-629, hsa-miR-629*, hsa-miR-630, hsa-miR-631, hsa-miR-632,hsa-miR-633, hsa-miR-634, hsa-miR-635, hsa-miR-636, hsa-miR-637,hsa-miR-638, hsa-miR-639, hsa-miR-640, hsa-miR-641, hsa-miR-642,hsa-miR-643, hsa-miR-644, hsa-miR-645, hsa-miR-646, hsa-miR-647,hsa-miR-648, hsa-miR-649, hsa-miR-650, hsa-miR-651, hsa-miR-652,hsa-miR-653, hsa-miR-654-3p, hsa-miR-654-5p, hsa-miR-655, hsa-miR-656,hsa-miR-657, hsa-miR-658, hsa-miR-659, hsa-miR-660, hsa-miR-661,hsa-miR-662, hsa-miR-663, hsa-miR-663b, hsa-miR-664, hsa-miR-664*,hsa-miR-665, hsa-miR-668, hsa-miR-671-3p, hsa-miR-671-5p, hsa-miR-675,hsa-miR-7, hsa-miR-708, hsa-miR-708*, hsa-miR-7-1*, hsa-miR-7-2*,hsa-miR-720, hsa-miR-744, hsa-miR-744*, hsa-miR-758, hsa-miR-760,hsa-miR-765, hsa-miR-766, hsa-miR-767-3p, hsa-miR-767-5p,hsa-miR-768-3p, hsa-miR-768-5p, hsa-miR-769-3p, hsa-miR-769-5p,hsa-miR-770-5p, hsa-miR-802, hsa-miR-873, hsa-miR-874, hsa-miR-875-3p,hsa-miR-875-5p, hsa-miR-876-3p, hsa-miR-876-5p, hsa-miR-877,hsa-miR-877*, hsa-miR-885-3p, hsa-miR-885-5p, hsa-miR-886-3p,hsa-miR-886-5p, hsa-miR-887, hsa-miR-888, hsa-miR-888*, hsa-miR-889,hsa-miR-890, hsa-miR-891a, hsa-miR-891b, hsa-miR-892a, hsa-miR-892b,hsa-miR-9, hsa-miR-9*, hsa-miR-920, hsa-miR-921, hsa-miR-922,hsa-miR-923, hsa-miR-924, hsa-miR-92a, hsa-miR-92a-1*, hsa-miR-92a-2*,hsa-miR-92b, hsa-miR-92b*, hsa-miR-93, hsa-miR-93*, hsa-miR-933,hsa-miR-934, hsa-miR-935, hsa-miR-936, hsa-miR-937, hsa-miR-938,hsa-miR-939, hsa-miR-940, hsa-miR-941, hsa-miR-942, hsa-miR-943,hsa-miR-944, hsa-miR-95, hsa-miR-96, hsa-miR-96*, hsa-miR-98,hsa-miR-99a, hsa-miR-99a*, hsa-miR-99b, and hsa-miR-99b*.

A miRNA inhibits the function of the mRNAs it targets and, as a result,inhibits expression of the polypeptides encoded by the mRNAs. Thus,blocking (partially or totally) the activity of the miRNA (e.g.,silencing the miRNA) can effectively induce, or restore, expression of apolypeptide whose expression is inhibited (derepress the polypeptide).In one embodiment, derepression of polypeptides encoded by mRNA targetsof a miRNA is accomplished by inhibiting the miRNA activity in cellsthrough any one of a variety of methods. For example, blocking theactivity of a miRNA can be accomplished by hybridization with a smallinterfering nucleic acid (e.g., antisense oligonucleotide, miRNA sponge,TuD RNA) that is complementary, or substantially complementary to, themiRNA, thereby blocking interaction of the miRNA with its target mRNA.As used herein, an small interfering nucleic acid that is substantiallycomplementary to a miRNA is one that is capable of hybridizing with amiRNA, and blocking the miRNA's activity. In some embodiments, an smallinterfering nucleic acid that is substantially complementary to a miRNAis an small interfering nucleic acid that is complementary with themiRNA at all but 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, or 18 bases, in some embodiments, an small interfering nucleic acidsequence that is substantially complementary to a miRNA, is an smallinterfering nucleic acid sequence that is complementary with the miRNAat, at least, one base.

A “miRNA Inhibitor” is an agent that blocks miRNA function, expressionand/or processing. For instance, these molecules include but are notlimited to microRNA specific antisense, microRNA sponges, tough decoyRNAs (TuD RNAs) and microRNA oligonucleotides (double-stranded, hairpin,short oligonucleotides) that inhibit miRNA interaction with a Droshacomplex. MicroRNA inhibitors can be expressed in cells from transgenesof a rAAV vector, as discussed above. MicroRNA sponges specificallyinhibit miRNAs through a complementary heptameric seed sequence (Ebert,M.S. Nature Methods, Epub Aug. 12, 2007;). In some embodiments, anentire family of miRNAs can be silenced using a single sponge sequence.TuD RNAs achieve efficient and long-term-suppression of specific miRNAsin mammalian cells (See, e.g., Takeshi Haraguchi, et. al., Nucleic AcidsResearch, 2009, Vol. 37, No. 6 e43, the contents of which relating toTuD RNAs are incorporated herein by reference). Other methods forsilencing miRNA function (derepression of miRNA targets) in cells willbe apparent to one of ordinary skill in the art.

In some embodiments, the cloning capacity of the recombinant RNA vectormay limited and a desired coding sequence may require the completereplacement of the virus's 4.8 kilobase genome. Large genes may,therefore, not be suitable for use in a standard recombinant AAV vector,in some cases. The skilled artisan will appreciate that options areavailable in the art for overcoming a limited coding capacity. Forexample, the AAV ITRs of two genomes can anneal to form head to tailconcatamers, almost doubling the capacity of the vector. Insertion ofsplice sites allows for the removal of the ITRs from the transcript.Other options for overcoming a limited cloning capacity will be apparentto the skilled artisan.

Somatic Transgenic Animal Models Produced Using rAAV-Based Gene Transfer

The disclosure also involves the production of somatic transgenic animalmodels of disease using recombinant Adeno-Associated Virus (rAAV) basedmethods. The methods are based, at least in part, on the observationthat AAV serotypes and variants thereof mediate efficient and stablegene transfer in a tissue specific manner in adult animals. The rAAVelements (capsid, promoter, transgene products) are combined to achievesomatic transgenic animal models that express a stable transgene in atime and tissue specific manner. The somatic transgenic animal producedby the methods of the disclosure can serve as useful models of humandisease, pathological state, and/or to characterize the effects of genefor which the function (e.g., tissue specific, disease role) is unknownor not fully understood. For example, an animal (e.g., mouse) can beinfected at a distinct developmental stage (e.g., age) with a rAAVcomprising a capsid having a specific tissue targeting capability (e.g.,liver, heart, pancreas, CNS) and a transgene having a tissue specificpromoter driving expression of a gene involved in disease. Uponinfection, the rAAV infects distinct cells of the target tissue andproduces the product of the transgene.

In some embodiments, the sequence of the coding region of a transgene ismodified. The modification may alter the function of the product encodedby the transgene. The effect of the modification can then be studied inviva by generating a somatic transgenic animal model using the methodsdisclosed herein. In some embodiments, modification of the sequence ofcoding region is a nonsense mutation that results in a fragment (e.g., atruncated version). In other cases, the modification is a missensemutation that results in an amino acid substitution. Other modificationsare possible and will be apparent to the skilled artisan.

In some embodiments, the transgene causes a pathological state. Atransgene that causes a pathological state is a gene whose product has arole in a disease or disorder causes the disease or disorder, makes theanimal susceptible to the disease or disorder) and/or may induce thedisease or disorder in the animal. The animal can then be observed toevaluate any number of aspects of the disease (e.g., progression,response to treatment, etc). These examples are not meant to belimiting, other aspects and examples are disclosed herein and describedin more detail below.

The disclosure in some aspects, provide methods for producing somatictransgenic animal models through the targeted destruction of specificcell types. For example, models of type 1 diabetes can be produced bythe targeted destruction of pancreatic Beta-islets. In other examples,the targeted destruction of specific cell types can be used to evaluatethe role of specific cell types on human disease. In this regard,transgenes that encode cellular toxins (e.g., diphtheria toxin A (DTA))or pro-apoptotic genes (NTR, Box, etc.) can be useful as transgenes forfunctional ablation of specific cell types. Other exemplary transgenes,whose products kill cells are embraced by the methods disclosed hereinand will be apparent to one of ordinary skill in the art.

The disclosure in some aspects, provides methods for producing somatictransgenic animal models to study the long-term effects ofover-expression or knockdown of genes. The long term over expression orknockdown (e.g., by shRNA, miRNA, miRNA inhibitor, etc.) of genes inspecific target tissues can disturb normal metabolic balance andestablish a pathological state, thereby producing an animal model of adisease, such as, for example, cancer. The disclosure in some aspects,provides methods for producing somatic transgenic animal models to studythe long-term effects of over-expression or knockdown of gene ofpotential oncogenes and other genes to study tumorigenesis and genefunction in the targeted tissues. Useful transgene products includeproteins that are known to be associated with cancer and smallinterfering nucleic acids inhibiting the expression of such proteins.Other suitable transgenes may be readily selected by one of skill in theart provided that they are useful for creating animal models oftissue-specific pathological state and/or disease.

Recombinant AAV Administration Methods

The rAAVs may be delivered to a subject in compositions according to anyappropriate methods known in the art. The rAAV, preferably suspended ina physiologically compatible carrier (i.e., in a composition), may beadministered to a subject, i.e. host animal, to such as a human, mouse,rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig,hamster, chicken, turkey, or a non-human primate (e.g., Macaque). Insome embodiments a host animal does not include a human.

Delivery of the rAAVs to a mammalian subject may be by, for example,intramuscular injection or by administration into the bloodstream of themammalian subject. Administration into the bloodstream may be byinjection into a vein, an artery, or any other vascular conduit. In someembodiments, the rAAVs ate administered into the bloodstream by way ofisolated limb perfusion, a technique well known in the surgical arts,the method essentially enabling the artisan to isolate a limb from thesystemic circulation prior to administration of the rAAV virions. Avariant of the isolated limb perfusion technique, described in U.S. Pat.No. 6,177,403, can also be employed by the skilled artisan to administerthe virions into the vasculature of an isolated limb to potentiallyenhance transduction into muscle cells or tissue. Moreover, in certaininstances, it may be desirable to deliver the virions to the CNS of asubject. By “CNS” is meant all cells and tissue of the brain and spinalcord of a vertebrate. Thus, the term includes, but is not limited to,neuronal cells, glial cells, astrocytes, cereobrospinal fluid (CSF),interstitial spaces, bone, cartilage and the like. Recombinant AAVs maybe delivered directly to the CNS or brain by injection into, e.g., theventricular region, as well as to the striatum (e.g., the caudatenucleus or putamen of the striatum), spinal cord and neuromuscularjunction, or cerebellar lobule, with a needle, catheter or relateddevice, using neurosurgical techniques known in the art, such as bystereotactic injection (see, e.g., Stein et al., J Virol 73:3424-3429,1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidson et al., Nat.Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther.11:2.315-2329, 2000). Recombinant AAVs may also be delivered indirectlyto the CNS, for example by intravenous injection.

The compositions of the disclosure may comprise an rAAV alone, or incombination with one or more other viruses (e.g., a second rAAV encodinghaving one or more different transgenes). In some embodiments, acomposition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more differentrAAVs each having one or more different transgenes.

Suitable carriers may be readily selected by one of skill in the art inview of the indication for which the rAAV is directed. For example, onesuitable carrier includes saline, which may be formulated with a varietyof buffering solutions (e.g., phosphate buffered to saline). Otherexemplary carriers include sterile saline, lactose, sucrose, calciumphosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, andwater. The selection of the carrier is not a limitation of the presentdisclosure.

Optionally, the compositions of the disclosure may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

The rAAVS are administered in sufficient amounts to transfect the cellsof a desired tissue and to provide sufficient levels of gene transferand expression without undue adverse effects. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the selected organ (e.g., intraportaldelivery to the liver), oral, inhalation (including intranasal andintratracheal delivery), intraocular, intravenous, intramuscular,subcutaneous, intradermal, intratumoral, and other parental routes ofadministration. Routes of administration may be combined, if desired.

The dose of rAAV virions required to achieve a particular “therapeuticeffect,” e.g., the units of dose in genome copies/per kilogram of bodyweight (GC/kg), will vary based on several factors including, but notlimited to: the route of rAAV virion administration, the level of geneor RNA expression required to achieve a therapeutic effect, the specificdisease or disorder being treated, and the stability of the gene or RNAproduct. One of skill in the art can readily determine a rAAV viriondose range to treat a patient having a particular disease or disorderbased on the aforementioned factors, as well as other factors that arewell known in the art.

An effective amount of an rAAV is an amount sufficient to target infectan animal, target a desired tissue. In some embodiments, an effectiveamount of an rAAV is an amount sufficient to produce a stable somatictransgenic animal model. The effective amount will depend primarily onfactors such as the species, age, weight, health of the subject, and thetissue to be targeted, and may thus vary among animal and tissue. Forexample, a effective amount of the rAAV is generally in the range offrom about 1 ml to about 100 ml of solution containing from about 10⁹ to10¹ genome copies. In some cases, a dosage between about 10¹¹ to 10¹²rAAV genome copies is appropriate. In certain embodiments, 10¹² rAAVgenome copies is effective to target heart, liver, and pancreas or CNStissues. In some cases, stable transgenic animals are produced bymultiple doses of an rAAV.

In some embodiments, rAAV compositions are formulated to reduceaggregation of AAV particles in the composition, particularly where highrAAV concentrations are present (e.g., ˜10¹³ GC/ml or more). Methods forreducing aggregation of rAAVs are well known in the art and, include,for example, addition of surfactants, pH adjustment, salt concentrationadjustment, etc. (See, Wright F R, et al., Molecular Therapy (2005) 12,171-178, the contents of which are incorporated herein by reference.)

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens.

Typically, these formulations may contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound in eachtherapeutically-useful composition may be prepared is such a way that asuitable dosage will be obtained in any given unit dose of the compound.Factors such as solubility, bioavailability, biological half-life, routeof administration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

In certain circumstances it will be desirable to deliver the rAAV-basedtherapeutic constructs in suitably formulated pharmaceuticalcompositions disclosed herein either subcutaneously,intraopancreatically, intranasally, parenterally, intravenously,intramuscularly, intrathecally, or orally, intraperitoneally, or byinhalation. In some embodiments, the administration modalities asdescribed in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (eachspecifically incorporated herein by reference in its entirety) may beused to deliver rAAVs. In some embodiments, a preferred mode ofadministration is by portal vein injection.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Dispersions may also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. In many cases the form issterile and fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art. For example, one dosage may be dissolvedin 1 ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the host. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual host.

Sterile injectable solutions are prepared by incorporating the activerAAV in the required amount in the appropriate solvent with various ofthe other ingredients enumerated herein, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The rAAV compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts, include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Supplementary active ingredients can also be incorporated into thecompositions. The phrase “pharmaceutically-acceptable” refers tomolecular entities and compositions that do not produce an allergic orsimilar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles,microspheres, lipid particles, vesicles, and the like, may be used forthe introduction of the compositions of the present disclosure intosuitable host cells. In particular, the rAAV vector delivered trangenesmay be formulated for delivery either encapsulated in a lipid particle,a liposome, vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids or therAAV constructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art. Recently, liposomes weredeveloped with improved serum stability and circulation half-times (U.S.Pat. No. 5,741,516). Further, various methods of liposome and liposomelike preparations as potential drug carriers have been described (U.S.Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures. In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, drugs, radiotherapeutic agents, viruses, transcriptionfactors and allosteric effectors into a variety of cultured cell linesand animals. In addition, several successful clinical trails examiningthe effectiveness of liposome-mediated drug delivery have beencompleted.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVS generally havediameters of from 25 nm can to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 .ANG., containing an aqueous solution in the core.

Alternatively, nanocapsule formulations of the rAAV may be used.Nanocapsules can generally entrap substances in a stable andreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use.

In addition to the methods of delivery described above, the followingtechniques are also contemplated as alternative methods of deliveringthe rAAV compositions to a host. Sonophoresis (ie., ultrasound) has beenused and described in U.S. Pat. No. 5,656,016 as a device for enhancingthe rate and efficacy of drug permeation into and through thecirculatory system. Other drug delivery alternatives contemplated areintraosseous injection (U.S. Pat. No. 5,779,708), microchip devices(U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al.,1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) andfeedback-controlled delivery (U.S. Pat. No. 5,697,899).

Kits and Related Compositions

The agents described herein may, in some embodiments, be assembled intopharmaceutical or diagnostic or research kits to facilitate their use intherapeutic, diagnostic or research applications. A kit may include oneor more containers housing the components of the disclosure andinstructions for use. Specifically, such kits may include one or more toagents described herein, along with instructions describing the intendedapplication and the proper use of these agents. In certain embodimentsagents in a kit may be in a pharmaceutical formulation and dosagesuitable for a particular application and for a method of administrationof the agents. Kits for research purposes may contain the components inappropriate concentrations or quantities for running variousexperiments.

The kit may be designed to facilitate use of the methods describedherein by researchers and can take many forms. Each of the compositionsof the kit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processableto an active form), for example, by the addition of a suitable solventor other species (for example, water or a cell culture medium), whichmay or may not be provided with the kit. As used herein, “instructions”can define a component of instruction and/or promotion, and typicallyinvolve written instructions on or associated with packaging of thedisclosure. Instructions also can include any oral or electronicinstructions provided in any manner such that a user will clearlyrecognize that the instructions are to be associated with the kit, forexample, audiovisual (e.g., videotape, DVD, etc,), Internet, and/orweb-based communications, etc. The written instructions may be in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which instructions canalso reflects approval by the agency of manufacture, use or sale foranimal administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. The agents may bein the form of a liquid, gel or solid (powder). The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container. The kit may have one ormore or all of the components required to administer the agents to ananimal, such as a syringe, topical application devices, or iv needle.tubing and bag, particularly in the case of the kits for producingspecific somatic animal models.

In some cases, the methods involve transfecting cells with totalcellular DNAs isolated from the tissues that potentially harbor proviralAAV genomes at very low abundance and supplementing with helper virusfunction (e.g., adenovirus) to trigger and/or boost AAV rep and cap genetranscription in the transfected cell. In some cases, RNA from thetransfected cells provides a template for RT-PCR amplification of cDNAand the detection of novel AAVs. In cases where cells are transfectedwith total cellular DNAs isolated from the tissues that potentiallyharbor proviral AAV genomes, it is often desirable to supplement thecells with factors that promote AAV gene transcription. For example, thecells may also be infected with a helper virus, such as an Adenovirus ora Herpes Virus. In a specific embodiment, the helper functions areprovided by an adenovirus. The adenovirus may be a wild-type adenovirus,and may be of human or non-human origin, preferably non-human primate(NHP) origin. Similarly adenoviruses known to infect non-human animals(e.g., chimpanzees, mouse) may also be employed in the methods of thedisclosure (See, e.g., U.S. Pat. No. 6,083,716). In addition towild-type adenoviruses, recombinant viruses or non-viral vectors (e.g.,plasmids, episomes, etc.) carrying the necessary helper functions may beutilized. Such recombinant viruses are known in the art and may beprepared according to published techniques. See, U.S. Pat. No. 5,871,982and U.S. Pat. No. 6,251,677, which describe a hybrid Ad/AAV virus. Avariety of adenovirus strains are available from the American TypeCulture Collection, Manassas, Va., or available by request from avariety of commercial and institutional sources. Further, the sequencesof many such strains are available from a variety of databasesincluding, e.g., PubMed and GenBank.

Cells may also be transfected with a vector (e.g., helper vector) whichprovides helper functions to the AAV. The vector providing helperfunctions may provide adenovirus functions, including, e.g., E1a, E1b,E2a, E4ORF6. The sequences of adenovirus gene providing these functionsmay be obtained from any known adenovirus serotype, such as serotypes 2,3, 4, 7, 12 and 40, and further including any of the presentlyidentified human types known in the art. Thus, in some embodiments, themethods involve transfecting the cell with a vector expressing one ormore genes necessary for AAV replication, AAV gene transcription, and/orAAV packaging.

In some cases, a novel isolated capsid gene can be used to construct andpackage recombinant AAV vectors, using methods well known in the art, todetermine functional characteristics associated with the novel capsidprotein encoded by the gene. For example, novel isolated capsid genescan be used to construct and package recombinant AAV (rAAV) vectorscomprising a reporter gene (e.g., B-Galactosidase, GFP, Luciferase,etc.). The rAAV vector can then be delivered to an animal (e.g., mouse)and the tissue targeting properties of the novel isolated capsid genecan be determined by examining the expression of the reporter gene invarious tissues (e.g., heart, liver, kidneys) of the animal. Othermethods for characterizing the novel isolated capsid genes are disclosedherein and still others are well known in the art.

The kit may have a variety of forms, such as a blister pouch, a shrinkwrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, ora similar pouch or tray form, with the accessories loosely packed withinthe pouch, one or more tubes, containers, a box or a bag. The kit may besterilized after the accessories are added, thereby allowing theindividual accessories in the container to be otherwise unwrapped. Thekits can be sterilized using any appropriate sterilization techniques,such as radiation sterilization, heat sterilization, or othersterilization methods known in the art. The kit may also include othercomponents, depending on the specific application, for example,containers, cell media, salts, buffers, reagents, syringes, needles, afabric, such as gauze, for applying or removing a disinfecting agent,disposable gloves, a support for the agents prior to administration etc.

The instructions included within the kit may involve methods fordetecting a latent AAV in a cell. In addition, kits of the disclosuremay include, instructions, a negative and/or positive control,containers, diluents and buffers for the sample, sample preparationtubes and a printed or electronic table of reference AAV sequence forsequence comparisons.

EXAMPLES Example 1

Development of novel AAV vectors for muscular and lung gene delivery: anengineered novel capsid variant of AAV9 for muscle and lung-directedtransgene expression

AAV-ClvD8 is an AAV9 variety with significantly reduced transcytosisproperty. Structural analysis of the ClvD8 capsid was performed usingnewly established AAV9 capsid structure as a template. This analysisrevealed that other than the alteration at position 647, the othermutated amino acid (a.a.) residues in Clv-D8 (compared with AAV9) arelocated on the AAV9 capsid protrusions that surround the icosahedral3-fold axis of AAV9 VP3 protein, which plays a role in receptor bindingand cellular transduction.

In an attempt to map the a.a. residual(s) responsible for peripheral(primarily liver) tissue transduction by IV delivery of rAAV9, singleand combinatory mutations for each of the a.a. on the AAV9 capsid thatare mutated in AAV-ClvD8 were produced. Corresponding luciferaseexpressing vectors were delivered into C57BL/6 mice by intravascular(IV), intranasal (IN) and intramuscular (IM) injections to compare theirluciferase transduction and vector genome biodistribution profiles withrAAV9 wild type (wt).

Using this approach, a novel capsid mutant of AAV9 was produced that isidentified herein as AAV9.HR that generated significantly lowerluciferase expression and vector genome abundance (VGA) in liver by allthree routes and local tissue-restricted expression and genomepersistence by IM and IN delivery. Other mutants that were producedusing this method are identified herein as AAV9H, AAV9I, AAV9R, andAAV9Y (as depicted in FIG. 1).

FIG. 2A-C depicts that Clv-D8, which is a chimpanzee-derivedAAV9-relative (Δ4 a.a.), has abolished transcytosis capability. FIG. 2Dshows that the blood clearance pattern is not change between AAV9 andCLv-D8, and other mutants of AAV9 (i.e., R533S, H527Y, and doubleR533S+H527Y compared with AAV9, referred to as AAV9R, AAV9H and AAV9HR,respectively) for IV or IM administration.

AAV9.HR (derived from CLv-D8, Δ2 a.a.) displayed a blood clearancepattern similar to that of rAAV9wt after IV, IM, and IN administrationsuggesting that its ability to cross vascular barrier from tissue tovessel was not impaired. Six-week old male C57BL/6 mice were injectedvia left tibialis anterior with AAV9 and its mutants (1.0 e11 GC/mouse,n=3). At specific time points (0-72 hrs), 5 μl of tail vein blood werecollected, diluted 1000-fold into PBS and then proceeded for qPCRanalysis. Standards were spiked with normal mouse blood (1000-folddilution) without treatment. See FIG. 3.

Luciferase assays were performed to evaluate the distribution of AAVs inmice after IM injection. It was found that although AAV9.HR can entervessel after IM injection, it is limited in its ability to enter majororgan, such as liver, by transcytosis. See FIG. 4. The AAV9.HR mediatedtransgene expression is essentially limited in injected muscle.

In another experiment, six-week old male C57BL/6 mice were injected viatail vein (1.0 e11 GC/mouse) with AAV9 and its mutant vectors encodingluciferase gene. Four weeks later, specific organs were harvested forqPCR and luciferase activity analysis. The results to are depicted inFIG. 5. It was found that AAV9.HR-mediated liver transgene expression ismuch lower than wtAAV9 after IV injection. (See also FIGS. 7 and 8.)

In another experiment, six-week old male C57BL/6 mice were injected viaintranasal delivery (1.0 e11 GC/mouse) with AAV9 and its mutant vectorsencoding luciferase gene. Four weeks later, specific organs wereharvested for qPCR and luciferase activity analysis. The results aredepicted in FIG. 6. It was found that AAV9.HR mediated transgeneexpression is limited in lung after IN delivery

Further assays were conducted in which four-week-old C57BL/6 mice wereinjected (IV, Retro-orbital injection, 1e12 GC/mouse,) with scAAV9wt.CB6.PI.EGFP (n=3) or scAAV9HR.CB6.PI.EGFP (n=3); three weeks afteradministration, organs were collected and gDNA were extracted for qPCRanalysis (Student's t-test (unpaired, two-tailed)). The results of theseassay are depicted in FIG. 9 and illustrate differences in thebiodistribution of AAV9 compared with its mutant vectors following IVadministration.

A comparison was also made of AAV9 and AAV9HR-mediated neurontransduction after IV (RO) delivery into D1 C57Bl6 mice at 4×10¹¹GC/mouse. Gene transfer was analyzed at 3 weeks post-injection.Comparable CNS gene transfer between these two vectors, as depicted inFIG. 10. The rAAV9.HR even leads to more neuron transduction (highration of transduced neuron/astrocyte) in certain areas (hippocampus,cortex etc.) It was also found that rAAV9.17IR leads to peripheralorgans detargeting, as depicted in FIG. 11.

A comparison was also made of AAV9 and AAV9HR-mediated biodistributionafter IV (RO) delivery into D1 C57Bl6 mice at 1×10¹² GC/mouse (a higherdose). Gene transfer was analyzed at 3 weeks post-injection. (FIG. 12.)Comparable vector genome abundance in CNS in adult animals between rAAV9and rAAV9HR, AAV9.HR leads to ˜135-fold less vector genome abundance andsignificantly diminished transgene expression in liver (data not shownhere) and other peripheral organs as compared to rAAV9

This work represents a significant advance in understanding vectorbiology of AAV9 and developing novel vectors with transductionefficiency similar to rAAV9 but improved safety profile for muscle andlung gene delivery and gene therapy of muscular, lung or otherdisorders.

Example 2

This example describes the use of AAV9.HR for CNS-directed gene therapyby intravenous (IV) injection.

A mouse model of Canavan disease was used in this study. Healthy control(SVT) and Canavan disease (CD) mice were administered a dose of 4×10¹¹genome copies (GC) of an rAAV vector containing the human aspartoacylase(AspA) gene (AAV9-FKzhAspA-OPT or AAV9.HR-FKzhAspA-OPT) at age p1 byintravenous injection. All untreated CD mice died by week 4. Enzymaticassays and western blot were performed on WT and treated CD mice at agep42. Administration of either construct prevented death of CD mice. FIG.13A shows no significant difference in enzymatic activity of AspA in thebrains of mice administered AAV9 or AAV9.HR. However, administration ofthe AAV9.HR construct did result in less off-target (e.g. liver tissue)expression of ASPA comparted to the AAV9 construct, as shown in FIG.13B.

FIG. 14A shows similar localization patterns of hAspA expression in thebrains of mice (age p42) after administration via AAV9 or AAV9.HR asmeasured by magnetic resonance imaging (MRI). Magnetic resonancespectroscopy (MRS) data demonstrates that hAspA expressed by bothconstructs is able to degrade N-acetyl-L-aspartic acid (NAA) (FIG. 14B).

FIG. 15 shows that mice administered the HR.FKzhAspA-Opt constructshowed no significant difference in growth (as measured by weight) toeither wild-type (WT) or AAV9.FKzhAspA-Opt treated mice. Untreated andvehicle-only treated CD mice died by 4 weeks of age.

Administration of HR.FKzhAspA-Opt to CD mice also improved motor andbehavioral phenotypes (FIGS. 16-18).

The results described above demonstrate that a CNS-targeted transgene(hASPA) delivered by an intravenously injected AAV9.HR vector isfunctionally expressed and localized in a similar manner toAAV9-delivered hAspA. However, the AAV.HR vecotor provides the advantageof lower off-target expression that AAV9. The results further show thatAAV9.HR-hAspA is effective in prolonging the life and improving motorphenotype in a mouse model of Canavan disease.

Example 3

This example describes the expression of secreted tumor suppressorproteins using AAV.HR vectors for cancer therapy.

Activated oncogenes can induce cellular senescence and/or apoptosis invitro and in vivo. Oncogene-induced senescence and/or apoptosis may be acellular mechanism to prevent proliferation of cells at risk forneoplastic transformation. One gene involved in this process is the BRAFoncogene, which is a serine-threonine protein kinase. BRAF is adownstream effector of RAS and signals through the MAP kinase pathway.Oncogenic BRAF-activating mutations (e.g. BRAFV600E) substantiallyincrease protein kinase activity, resulting in constitutive ERKsignaling. These activating BRAF mutations are found in 70% ofmelanomas, 15% of colorectal cancers, 30% of ovarian cancers and 40% ofpapillary thyroid carcinomas. Several factors are involved inBRAF-mediated senescence/apoptosis (Table 1).

TABLE 1 Factors involved in BRAF-mediated Senescence/Apoptosis BIN1ILIR1 RAP1GAP TP53 SMARCB1 BNIP3L HSPA9B PEA15 NF2 BUB1 IGFBP7 DMTF1FOXA1 MEN1 HIRA IRF1 FBXO31

Previous studies have shown IGFBP7 suppresses growth ofBRAFV600E-positive tumors in mouse xenografts. Furthermore, IGFBP7suppresses melanoma metastasis and enhances survival in mice with tumorxenografts. Another protein that has potential use as a cancertherapeutic is Sushi repeat-containing protein (SRPX), which has beenshown to be down-regulated in many human lung cancers. SRPX is predictedto be a secreted protein.

FIG. 19A shows that AAV9.HR-IGFBP7 has tumor suppression activitycomparable to IGFBP delivered by WT AAV9. FIG. 19B shows thedose-dependent effect of AAV.HR-IGFBP7 on tumor suppression. The tumorsuppressing activity of SRPX was also demonstrated. FIG. 20A shows theconcentration-dependent effect of AAV9.HR-SRPX delivery on tumor size inBalb/c Nu/Nu mice. FIG. 20B shows dose-dependent expression of SRPXdelivered by AAV9.HR vector. Due to the reduced transcytosis propertiesexhibited by the AAV9.HR vector, transgene delivery is reversible,whereas delivery by WT AAV9 vector persists even after removal of theinjected tissue (FIG. 21). FIG. 22 demonstrates that AAV9.HR vectorgenomes do not migrate away from the tissue into which they are todelivered.

The toxicity of transgene delivery by AAV.HR vectors was assessed bymeasurement of alanine transaminase (ALT) and aspartate aminotransferase(AST) (FIG. 23-25). AAV9.HR exhibits a similar toxicity profile to WTAAV9 at several dosages. These results indicate that AAV.HR is a safeand efficient vector for muscle delivering of the secreted tumorsuppressor proteins as cancer therapeutics.

SEQUENCES AAV9 CAPSID >SEQ ID NO: 1MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLT RNLCLVD8 CAPS ID >SEQ ID NO: 2MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLHYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASYKEGEDSFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQTLIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYL TRNLAAV9H CAPSID >SEQ ID NO: 3MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASYKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLT RNLAAV9I CAPSID >SEQ ID NO: 4MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQTLIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYL TRNLAAV9R CAPSID >SEQ ID NO: 5MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDSFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLT RNLAAV9Y CAPS ID >SEQ ID NO: 6MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLHYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLT RNLAAV9.HR CAPSID >SEQ ID NO: 7MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASYKEGEDSFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLT RNLNUCLEIC ACID ENCODING AAV9 CAPSID >SEQ ID NO: 8ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGNUCLEIC ACID ENCODING CLVD8 CAPSID >SEQ ID NO: 9ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGCACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCTACAAAGAAGGAGAGGACAGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGACCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGNUCLEIC ACID ENCODING AAV9H CAPSID >SEQ ID NO: 10ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCTACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGNUCLEIC ACID ENCODING AAV9I CAPSID >SEQ ID NO: 11ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGACCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGNUCLEIC ACID ENCODING AAV9R CAPSID >SEQ ID NO: 12ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTGATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACGCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACAGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGNUCLEIC ACID ENCODING AAV9Y CAPSID >SEQ ID NO: 13ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGCACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAACGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGNUCLEIC ACID ENCODING AAV9.HR CAPSID >SEQ ID NO: 14ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCTACAAAGAAGGAGAGGACAGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGProtein Alignment Consensus Sequence from FIG. 1 >SEQ ID NO: 15MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRIGTRYLT RNL

This disclosure is not limited in its application to the details ofconstruction and the arrangement of components set forth in thisdescription or illustrated in the drawings. The disclosure is capable ofother embodiments and of being practiced or of being carried out invarious ways. Also, the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting. Theuse of “including,” “comprising,” or “having,” “containing,”“involving,” and variations thereof herein, is meant to encompass theitems listed thereafter and equivalents thereof as well as additionalitems.

Having thus described several aspects of at least one embodiment of thisdisclosure, it is to be appreciated various alterations, modifications,and improvements will readily occur to to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe disclosure. Accordingly, the foregoing description and drawings areby way of example only.

1.-3. (canceled)
 4. A host cell containing a nucleic acid that comprisesa coding sequence selected from the group consisting of: SEQ ID NOs:10-14 that is operably linked to a promoter.
 5. A composition comprisingthe host cell of claim 4 and a sterile cell culture medium.
 6. Acomposition comprising the host cell of claim 5 and a cryopreservative.7.-38. (canceled)
 39. A recombinant AAV (rAAV) comprising an AAV capsidprotein having an amino acid sequence of SEQ ID NO: 1 with one or moreamino acid mutations selected from the group consisting of Y445H, H527Y,I647T, and R533S.
 40. The rAAV of claim 39 further comprising at leastone transgene.
 41. A method for delivering a transgene to a subjectcomprising administering the rAAV of claim 40 to a subject, wherein therAAV infects cells of a muscle tissue of the subject.
 42. The method ofclaim 41, wherein the at least one transgene encodes a protein or asmall interfering nucleic acid.
 43. The method of claim of claim 41,wherein the muscle tissue is skeletal muscle or heart tissue.
 44. Themethod of claim 41, wherein the rAAV is administered intravenously,intravascularly, or intramuscularly.
 45. The rAAV of claim 39, whereinthe one or more amino acid mutations are H527Y and R533S.
 46. The rAAVof claim 45 further comprising at least one transgene.
 47. A method fordelivering a transgene to a subject comprising administering the rAAV ofclaim 46 to a subject, wherein the rAAV infects cells of a muscle tissueof the subject.
 48. The method of claim 47, wherein the at least onetransgene encodes a protein or a small interfering nucleic acid.
 49. Themethod of claim of claim 47, wherein the muscle tissue is skeletalmuscle or heart tissue.
 50. The method of claim 47, wherein the rAAV isadministered intravenously, intravascularly, or intramuscularly.
 51. Anucleic acid encoding the rAAV of claim
 39. 52. A nucleic acid encodingthe rAAV of claim
 45. 53. A recombinant AAV (rAAV) comprising an AAVcapsid protein having one or more amino acid mutations at positionscorresponding to Y445H, H527Y, I647T, and R533S of SEQ ID NO:
 1. 54. TherAAV of claim 54, wherein the AAV capsid protein has amino acidmutations at positions corresponding to H527Y and R533S of SEQ ID NO: 1.55. A method for delivering a transgene to a subject comprisingadministering the rAAV of claim 54 to a subject, wherein the rAAVfurther comprises the transgene, and wherein the rAAV infects cells of amuscle tissue of the subject.